methods cell biology 2015

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Methods in Cell Biology 2015Johannes A. Schmid

Internet: www.meduniwien.ac.atuser!ohannes.schmid

some contents contri"uted "y:

#r. $u%as Mach

Institut &'r Angewandte (eneti% und )ell"iologie *+ni,. &. Boden%ultur

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-,er,iew o& o/ics

scienti&ic strategies and assay systems

cell culture

la"elling and trans&ection o& cells

analysis o& cellular com/onents

analysis o& molecular interactions

&luorescence measurements

microsco/y

&low analysis &luorescence acti,ated cell sorting* ACS3

analysis o& ,arious cellular /rocesses /roli&eration* a/o/tosis..3



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#etails o& the lecture

1. Scienti&ic strategies2. cell culture4. la"elling and trans&ection o& cells

a3 radioacti,e and chemical la"elling"3 trans&ections: o,ere/ression o& genes and gene su//ressionc3 re/orter gene assays

6. gene7su//ression si89A7technologies35. analysis o& #9A and /roteins electro/horesis and "lotting3. su"cellular &ractionation centri&ugations;3<. methods to detect macromolecular interactions

a3 =east 17 und 27hy"rid systems"3 co7immuno/reci/itationsc3 &luorescence resonance energy trans&er 8>3

?. methods o& &luorescence measurements@. realtime7C8 10. transmitted light microsco/y and contrast /rinci/les11. &luorescence microsco/y12. con&ocal laser scanning microsco/y14. &low analysis ACS316. analysis o& ,arious cellular /rocesses /roli&eration* a/o/tosis..315. methods to in,estigate ,esicular trans/ort /rocesses

9odes o& regulation in cellular systems

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#9A

/re7m89A m89A

degradation

degradation

/osttranslationalmodi&ication

translation

transcri/tion

trans/ort

s/licing

transcri/tion

&actor

trans/ortm89A

/re7micro789A micro789A

/rocessingmicro789A

acti,ation

signal transduction

/oly7u"iuitination

nucleus

>8

cell mem"ranerece/torsligands

(olgi

trans/ort

/rotein

trans/ort

signaltransduction

oligomeriation

modi&ied/rotein



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(eneral scienti&ic strategies

descri/ti,e strategies: the whole system cell* organism3 iso"ser,ed without in&luencing it

ad,antage: /hysiological states are not altereddisad,antage: it is di&&icult to elucidate cause7e&&ect relationshi/s

mechanistic mani/ulating3 strategies: ,arious &actors are %e/tconstant* while others are altered on /ur/ose D the change inthe whole system is monitored.

ad,antage : cause7e&&ect relationshi/s can "e monitored or

detecteddisad,antage : the /hysiological steady state is altered andin&luenced. 8esults might "e arte&acts o& the measurementsystem.

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>/erimental Systems I

In ,itro* "iochemical systems: in,estigation in a solutione.g. enyme reactions3: many /arameters can "e &iedtem/erature* /E* "u&&er com/osition;3.>am/le: enyme acti,ity assay* >MSA ;

In ,itro* cell "iological systems: including cellular structures

e.g. in ,itro7transcri/tiontranslation with mem"ranecom/onents* in ,itro7&usion o& endosomes* nuclear im/ortassays with isolated nuclei etc. >/erimental conditionsha,e to "e set in a way that cellular structures are notdamaged e.g. isotonic "u&&er* /hysiological /E;.3F reaction/artners can "e in&luenced widely e.g. anti"odies can "eadded;3



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>/erimental Systems II

cell culture systems7 immortalied cell lines: unlimited /assages

7 /rimary cells: limited /assage num"er* morecom/licated cell culture e.g. coated /lates;3

7 co7culture o& di&&erent cell ty/es:e.g. %eratinocytes G &i"ro"lasts in a collagen matri.

xenograft systems:- cells are in!ected into immuno7com/romied micenude mice* SCI# mice3F e.g. su"cutaneously7 tissue recom"ination systems e.g. /rostate e/ithelialcells with mesenchymal cells in!ected into the renalca/sule o& SCI# mice3

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Organ culturese.g. s%in sheets*"rain slices;3

Organ perfusions

>/erimental Systems III



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Animal Experiments:

Just the whole organism /ro,ides the &ull com/le "iological system that is

rele,ant &or most "iomedical research to/ics. he whole organism includes

su/erordinated systems such as the ner,ous system* the "lood circulation*

the endocrine system and so on. he cells are in their normal organ

en,ironmentF cellular communications are intact;. D thus the highest

/ossi"le /hysiological state can "e achie,ed. Eowe,er* s/eci&ic com/onents

o& the system e.g. certain cells3 are not easily accessi"le D and s/eci&ic

e/erimental mani/ulations e.g. o& s/eci&ic cells without side e&&ects3 are

o&ten di&&icult3. Cause7e&&ect relationshi/s are o&ten di&&icult to elucidate D

and there is a "ig H"lac% "o due to the com/leity o& the system.

It has to "e considered that results o"tained with animals such as mice o&ten

cannot "e trans&erred to human "eings des/ite the highly /hysiological

system.

>/erimental Systems I

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>/erimental Systems

Special case: Transgene and knock-out animals :S/eci&ic elimination o& genes %noc%7out3 or incor/oration o& genestransgenes* %noc%7in3 allow a "etter elucidation o& cause7e&&ect relationshi/s.Eowe,er* classical %noc%7outs eliminating a gene in all cells3 D is o&tenem"ryonically lethal D and does not allow conclusions &or its &unction in theadult animal e.g. mouse3. ice ,ersa* it can ha//en that there is no/henoty/e i& the &unction o& the gene is ta%en o,er "y another gene3. Knoc%7ins can "e uite arti&icial as well i& the transgene is e/ressed at higher le,els

with strong /romoters3. Modern a//roaches o&ten aim &or Hconditional %noc%7outs or %noc%7ins: In most cases the Cre7recom"inase lo system is used&or that /ur/ose:Conditional %noc%7out: the gene or a crucial eon3 is /laced "etween losites D Cre recom"inase which can "e e/ressed in s/eci&ic organs or cells "yorgan s/eci&ic /romoters3 cuts out the gene D thus the gene is deleted !ust ina certain organ or cell ty/eF using induci"le Cre* this system allows genedeletion at a de&ined time /oint e.g. when the animal is adult3.Conditional %noc%7in: the gene is /laced "ehind a Sto/7cassette* which is&lan%ed "y lo sites. Lithout Cre acti,ity* the gene is not e/ressed* with Creacti,ity organ s/eci&ic3* the Sto/ cassette is ecised and the gene is e/ressed



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Conditional transgene mouse models with the

Cre lo system

Cre recom"inase cuts outseuences "etween lo sitesor in,erts seuences "etweenin,erted lo sites3

Cre e/ression can "erendered cell7ty/e or organ7s/eci&ic using cell7ty/e s/eci&ic/romoters dri,ing Cree/ression s/atial control3

Cre e/ression can "e madeinduci"le "y using chimeras o&Cre with mutated estrogenrece/tor domainstem/oral control:e.g. Cre7>823

>ndogenous genes can "e&lan%ed "y lo sites usingrecom"ination techniues De.g. a&&ecting essential eons3 conditional %noc%7out

(enes can "e o,ere/ressedconditionally "y inserting ane/ression construct headed

"y a lo7&lan%ed HSto/cassette* which is cut out "yCre recom"inase

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1. Cre mouse strain2. lox-mouse strain

>am/les &or conditional mouse models

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Crem>8 m>8

mE! : mutated estrogen rece/tor res/onds to tamoi&en as ligandF withoutthe ligand it %ee/s the Cre in the cytosol inacti,eF u/on addition o& tamoi&en* the nuclear localiation seuences o& m>8 "ecome acti,e leading

to translocation o& the chimera into the nucleus* where Cre recom"inase caneert its &unction on lo7&lan%ed #9A seuences3.

cell7ty/e s/eci&ic/romoter

Cre7>8 2

Sto/ (ene o& interest(ene o& interest

lo lo good /romotere.g. /CA((S3

Crem>8 m>8

amam

Crem>8 m>8

amam

conditional knock-out conditional transgene



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S/eci&ic cell a"lation or cell la"eling

in transgenic mice

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Sto/ #8 #i/hteria toin rece/tor3

good /romotere.g. /CA((S3

lo lo

cross7"reeding with a cell7ty/e s/eci&ic Crestrain

#8 is e/ressed only in s/eci&ic cell ty/es

in!ection o& di/hteria toin leads to s/eci&ic

%illing o& these cells

Sto/ >= enhanced yellow &luorescent /rotein3

good /romotere.g. /CA((S3

lo lo

&luorescent la"eling o& a s/eci&ic cell ty/e o& interest

(enome >diting &or generating transgeneanimals

9o,el methods to edit genes directly in the genome* e.g. usingC8IS8Cas@ technology or )n7&inger nucleases* or A$>9sallow &aster and e,en multi/leing ty/e mani/ulations e.g.targeting 5 genes simultaneously3

(enome editing is then usually done in >S7cells* which are

su"seuently in!ected into "lastocysts.

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Cell Culture Methods cell culture o& immortalied cells

cell culture o& /rimary cells o&ten !ust a &ew /assages3

culture o& /olaried cells

co7culture o& di&&erent cell ty/es

/rimary &i"ro"lasts&rom s%in dermis3

trans&ormed&i"ro"lasts

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Lhat you need &or cell culture

Incu"ator 4<NC3 with C-27su//ly and humidi&ication

Sterile "ench $aminar low: alaminar &low o& &iltered air %ee/sthe "ench sterile3 D has to "eswitched on a//ro. 10 min

"e&ore using it* the &ilter has to"e chec%ed &rom time to timearticle Measurement3

in,erted cell culture microsco/e6* 10* 20 o"!ecti,es3

$27"iosa&ety e.g. &or ,irus7wor%3: not only incoming air is&iltered* "ut also the air thatlea,es the laminar &low.



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mammalian cells need media with ,itamins* aminoacids* hormones and growth &actors

Serum* mostly &etal cal& serum CS3* is the source o&growth &actors such as (* >(;3

Common com/osition7 #M>M #ul"eccoOs Modi&ied >ssential Medium37 10P CS7 2 mM (lutamine unsta"le amino acid* has to "e addedagain* i& the medium is older than a//. wee%s3

7 enicillin 100 uml37 Stre/tomycin 100 Qgml3

Culture Media

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has high concentrations o& growth &actorse.g.: >(* e/idermal growth &actorF (*&i"ro"last growth &actorF I(* insulin7li%egrowth &actor3

low amount o& anti"odies → com/ati"lewith cells o& other s/ecies

com/lement system is inacti,ated "y aheat shoc% 40 min.* 5 °C3

disad,antage o& CS: e/ensi,e ≈ 100 R/er $iter3

might contain contaminants such astetracyclin im/ortant i& you use a et7induci"le cell culture system /urchaseguaranteed et7&ree CS3

Alternati,e sources o& serum: normal cal&serum* horse serum;

etal Cal& Serum CS3 S/ecial (rowthactors

9er,e7 (rowth actor9(3:&or neuronal cells

Ee/atocyte7(rowth

actor E(3: induces celldi,ision o& he/atocytes

Keratinocyte7(rowthactor K(3: &or cultureo& s%in e/ithelial cells%eratinocytes3



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adherent cells ,ersus sus/ension cells

cell culture dishes: usually made hydro/hiliccharged grou/s3 D su&&icient &or most cell ty/es

collagenF (elatine denatured collagen3sometimes necessary &or good attachment e.g. &or/rimary endothelial cells3

Com/onents o& etracellular matri:

i"ronectin* $aminin D o&ten "etter than collagen.

s/ecial case: Heeder7cells irradiated3 D or co7culture o& cells e.g. &i"ro"lasts in a collagen matriwith %eratinocytes on to/3

Adhesion &actors

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Bacteria: might "e a /ro"lem* whenthey are resistant against the anti"ioticsthat are used mostly enStre/3 then you ha,e to use other anti"ioticse.g. %anamycin3

Myco/lasm: /rocaryotes o& an anciente,olutionary stage* which do not ha,e anormal "acterial cell wall there&orethey are resistant against enicillin3can "e eliminated with Kanamycin3

=east: rare might occur when yeastand mammalian cell culture are notstrictly se/arated3

ungi: uite rare

Contaminations in cell culture



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#9A7staining with #AI or Eoechst 4425?

Alternati,e:C87 detection o& Myco/lasm7#9Acommercial %its are a,aila"le3

no Myco/lasm

Myco/lasm

ests &or Myco/lasm7Contamination

+nnoticed myco/lasm contaminations can screw u/ e/erimental results

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Cell culture o& /olaried cells

a/ical side

lateral side

"asal mem"rane

the cells ha,e to "uild thetight !unctions &or "uilding u/the /olarity.

-&ten they are culti,ated on

&ilters* where the two sidesare accessi"le.

tight !unction



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Methods to in,estigate /olaried cells

Model systems

1. sim/le cell culture: !ust one side o& the cell is accessi"le.

2. cell culture on micro/orous mem"ranes: "oth sides aree/erimentally accessi"le* the esta"lishment o& a tight/olaried monolayer can "e chec%ed "e measuring theelectrical resistance "etween the two sides.

4. -rgan cultures: e.g. $i,ing S%in >ui,alent

6. -rgan /er&usion: e.g. /er&usion o& isolated rat li,er

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Culture o& /olaried cells on mem"ranes

Measurement o& electricalresistance



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Culti,ation o& /olaried cells on electrode

cham"ers

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Culti,ation o& /olaried cells on electrodecham"ers



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-rganoty/ic culture: >am/le: S%in >ui,alent

Keratinocytes cells o& the u//er layer o& s%in* the e/idermis3 areseeded onto a collagen matri* which contains &i"ro"lasts dermiscells3. As soon as they "uild a monolayer* they are ele,ated to thesur&ace o& the medium with their u//er side e/osed to air3 D thisinduces cell di&&erentiation and the &ormation o& a /seudo7e/idermiswith se,eral cell layers.

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-rgan er&usion

olaried cells such as he/atocytescan "e maintained in their organarchitecture maintaining their/olarity. he organ is /er&usedusing glas ca/illaries lin%ed to thenormal "lood ,essels the su//lythe organ with "lood and

nutrientsoygen3. A "u&&er 4<°C*/ercolated with -23 containing thenutrients can "e /er&used throughthe li,er. Mar%er su"stances can"e a//lied and their trans/ort&rom the "lood side "asolateral3to the "ile side a/ical3 can "edetermined.

Perfusionsdruck (cm H 2O)

V. porta

Kanüle

Peristaltikpumpe

V. cava Kanüle

Thermosensor

LEBER

Gallengangskanüle

FraktionskollektorComputer

Temperatur-

Messung

Schreiber

93% O2

7% CO2

Wasserbad

Gas-Befeuchtung

Perfusionspuffer

/er&usion /ressure cm E2-3

$i,er

"ile canula

. /orta

. ca,a canula

tem/erature

recording

gas humidi&ication

&raction collector

um/

Later Bath

er&usion Bu&&er



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>am/le o& an e/eriment with /olaried cells

ransmigration o& $eu%ocytes through a Monolayer o& /olariedendothelial cells

$eu%ocytes are la"eled with a&luorescent mar%er e.g. CS>

D an ester* which is turned&luorescent !ust a&ter u/ta%einto cells due to esterases3

Cells are seed onto a layer o& endothelial cells e.g. a&ter acti,ating the

endothelial cells with in&lammatory cyto%ines D which leads to thesynthesis o& adhesion molecules on the sur&ace o& the endothelialcells3. Adhesion o& leu%ocytes leads to transmigration into the lowercham"er. he etent o& the transmigration can "e determined "y lysingthe cells and uanti&ying the &luorescence or "y counting the&luorescent cells3

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Methods &or la"eling o& cellular com/onents

8adioacti,e la"eling: e.g. with radioacti,e amino acids 45S7Methionin;3* which are incor/orated into newly synthesied/roteins. ulsechase e/eriments can gi,e insights into hal& li&e*trans/ort. rocessing; o& /roteins.

Internalisation o& high molecular weight mar%ers loading o&endosomes and lysosomes3.

$a"eling o& cell sur&ace /roteins e.g. "y "iotinylation with cell7im/ermea"le reacti,e "iotin com/ounds3.

Addition o& cell7/ermea"le la"eled su"stances* which integrate intos/eci&ic structures e.g. (olgi7s/eci&ic &luorescent li/ids3

rans&ection o& cells with e/ression /lasmids

rotein7ransduction cell7/ermea"le /e/tides3

Micro7In!ection o& su"stances.



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Amino acids: 45STMethionine* 4ET$eucin

Monosaccharides: 4ETMannose* 4ET(lucosamine

hos/hate: 42T /hos/horic acid Alternati,e: 44: lower radioacti,ity

Sul&ate: 45STSul&uric acid

atty acids: 4ETalmitinic acid

8adioacti,e $a"eling o& Biomolecules

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8adionuclides



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usually 45STMethionine la"eling

ad,antage: high s/eci&ic radioacti,ity

disad,antage: relati,ely rare amino acid chec% &irst*how many methionine are in the /roteinU e.g.+"iuitin: only 1 Methionine* "ut @ $eucines3

reuires Methionine7&ree culture medium &or de/letiono& endogenous stores "y /re7incu"ation

addition o& dialyed CS3

Meta"olic la"eling with amino acids

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4ETMannose or 4ET(lucosamine

Acti,ated in the cytosol "y cou/ling to nucleotides →

(#7Mannose* +#797Acetyl7(lucosamine +#7(lc9Ac3

reuires (lucose7/oor culture medium

Addition o& alternati,e energy sources

(lutamine* yru,ate3

Meta"olic $a"eling with Monosaccharides



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/reincu"ation without mar%er de/letion o&endogenous stores3

Addition o& the la"eled su"stance → VulseV 5 min 71 h or longer3

Sto/ o& the VulseV "y addition o& an ecess o&unla"eled com/ound

&urther incu"ation → VChaseV min 7 6? h3

Sto/ o& culture → Analyses

ulse7Chase7>/eriments

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i"ro"lasts o& /atients* were la"eled with 45S7methionine:

ulse: 1 h at 4<°CChase: < h at 1@°C and 4<°C 1@°C inhi"its trans/ort &rom trans7(olgi tolate endosomes/re7lysosomes3 Immuno/rec. and autoradiogra/hy

>am/le &or a ulse7Chase7>/eriment



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direct: modi&ication o& yrosine with 125JT:7 chemical oidation: Chloramine7 oidies 9a125Jand leads to iodination o& yrosine7 enymatic: $acto/eroidase: oidies 9a125J in/resence o& E2-2.

indirect: Modi&ication o& amino grou/s $ysine* 97erminus3 with 125JT7la"eled Bolton7Eunter78eagen

97succinimidyl 4767hydroy 57125

JTiodo/henyl37/ro/ionate3

Iodination o& roteins $a"eling with 125J3

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Eigh a&&inity "inding /artner o& A,idin andStre/ta,idin easy /uri&ication anddetection "y "eads* coated >$ISA7lates;3

Stre/ta,idin has 6 "inding sites &or "iotincom/lees can "e &ormed with "i,alent"iotin7lin%ersU3 signal am/li&ication is

/ossi"le ABC: a,idin7"iotin7com/lees3

Most commonly used method to la"el cellsur&ace /roteins

Biotinylation o& amino grou/s with Biotin7Eydroysuccinimid7>sters or o& cysteinesSE7grou/3 "y Maleimide7deri,ates

Biotinylation



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$a"eling o& >ndosomes* $ysosomes

By high molecular weight com/ounds* which are easilydetecta"le* not /ermea"le &or the cyto/lasmic mem"rane andta%en u/ "y endocytosis>am/les:a3 uns/eci&ic internaliation:7 IC7#etran luorescein7la"eled37 >nymes eroidase;3"3 S/eci&ically "y rece/tors

ulseChase conditions can "e used to la"eled s/eci&ically earlyor late endosomes or lysosomes e.g. using tem/erature"loc%s....3

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rans&ections

+sually designates the incor/oration o& #9A into mammalian cells. #9A /resentin &orm o& /lasmids.

ransient rans&ection: /lasmid remains outside o& the genome and is slowlylost degradation* dilution "y cell di,ision3* ece/tion: e/isomal re/lication De.g. S607lasmids in C-S7cells3. he trans&ection e&&iciency ,aries D "ut canreach close to 100P

Sta"le rans&ection: integration o& &oreign #9A into the genome >&&iciency:

usually "elow 0.1P3. Isolation o& sta"ly trans&ected clones reuires selectiongenes &or anti"iotic resistance* e.g. /uromycin* (61?;3. lasmids are usually

linearied "e&ore trans&ection to increase the /ossi"ility o& correct integration.



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>am/le &or a mammalian e/ression /lasmid

CM7romoter

Multi/leCloning Site

target gene

/oly7adenylation signalsuited &or lineariation

S607romoter

selction gene"acterialselection withKanamycin*mammalian7selection with(61?3

/oly7adenylation signal

8e/lication origins no shown3

re/orter gene

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Selection mar%ers &or sta"le trans&ections

Aminoglycoside7hos/hotrans&erase: 8esistance against 9eomycin"acteria3 und (61? mammalian cells3. Sele%tion with (61? ta%esuite long a// 2 wee%s3. Sur,i,ing colonies are isolated and &urthercultures under selection /ressure.

Eygromycin7hos/hotrans&erase

uromycin7Acetyltrans&erase

#ihydro&olat78eductase #E83: Selection with MethotreatF allowsam/li&ications o& the target gene.

(7usion /roteins: &luorescencecan "e used &or selection: eam/le:>(797κ B e/ressing CE-7cells



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Chemical rans&ection Methods

"#A-Calcium precipitates: at eact /E andCa2G7concentration: Eigh e&&iciencies with2@47cells @0P and more3* e/ression le,elsare usually moderate.

$iposome mediated transfection: %ationicli/ids which "ind the negati,ely charged #9A*and which are ta%en u/ as li/osomesWuite high trans&ection e&&iciencies "ut alsosometimes toic e&&ects and /otentially

arte&acts "y high e/ression le,els oralteration o& cellular mem"ranes. e.g.$i/o&ectamine* ugene...3

Ca2G

Ca2G

Ca2G

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Buffers

• HeBS-Buffer (Hepes buffered saline)

8 g NaCl - 280 mM final concentration

0.2 g Na2HPO4.7H2O (or 0.107 g anhydrous) - 1.5 mM

6.5 g Hepes (Sigma H-7006) (or 5.96 g of free acid) - 50 mM

400 ml A.dest.

Adjust the pH to exactly 7.05 (calibrate pH-meter with pH 4.01 and pH 7.00 buffers before). Add A.dest. to 500 ml,

filter through 0.2 µm filters and store in aliquots at -20°C (not longer than 6 months). Thawed aliquots shouldn't be

frozen again.

• CaCl2: 29.4 g CaCl2.2H2O (MW=147) in 100 ml A.dest (final conc.: 2 M) Filter through 0.2 µm filters and store

aliquoted at -20°C.

• Chloroquine (optional): chloroquine. 2H2O (Sigma C-6628): 12.9 mg/ml in PBS (conc.: 25 mM). Filter through

0.2 µm filters and store at -20°C.

Procedure (amounts are given for 6-wells):

1. Seed cells (about 500 000 cells per 6-well = per 10 cm2 ) one day before the transfection (in DMEM/10% FCS)2. (Optional: 1 h before transfection, exchange the medium for medium containing 25µM chloroquine)

3. Thaw HeBS and CaCl2 at room temperature

4. For each transfection prepare aliquots of 71 µl HeBS

5. Prepare the DNA/ CaCl2-Mix: 4 µg DNA (total) in 62 µl A.dest. + 9 µl CaCl2

6. Add the DNA/ CaCl2-Mix drop-wise to the HeBS aliquots (by screwing the Gilson pipette) and slightly mix after

each drop. Incubate for 2 - 3 min at R.T. to form the DNA-precipitate (not longer).

7. Add the DNA-precipitate drop-wise to the cells (by screwing the Gilson pipette and moving it to cover the whole

surface of the cell culture; don't swirl the dish).

8. Carefully transfer the dish back to the incubator. Incubate for 24 h (or in the presence of chloroquine: for 10 h)

and exchange the medium afterwards. (The transfection is in the presence of FCS!). The efficiency of transfection is

in the range of 70-90% for 293 cells. Harvest the cells after 48 h. The protocol is adapted from Neil Perkins who

adapted it from Gary Nolan in 1995

(See web site: http://www.stanford.edu/group/nolan/ or CP in Mol.Biol. 9.1 and 9.11.2-3)

rotocoll: Calcium7rans&ection



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$i/o&ectamine2000 7 Standard conditions

rans&ection in /resence o& serum e.g. 26h D 6?h3

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Chemical rans&ection Methodes II

"endrimeres: charged olymeres that "ind#9A e.g. Su/er&ect Wiagen3

com/lees with "EAE-"extran /olycationic

#etran3

poly-et%ylene-imine &E'(:



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hysical rans&ection Methods

>lectro/oration: common withsus/ension cells*reuires much #9A S/ecial case: Amaa79ucleo&ection now $ona3

article7gun (ene (unX3:#9A on gold /articles shot "y/ressure onto cells e.g.neurons in "rain slices3.

Micro7In!ection o& #9A: intosingle cells !ust limited cellnum"er can "e targeted3.

electro7cu,ette

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iral rans&ection Methods

Adeno)iruses* !etro)iruses*$enti)iruses:

iruses de,elo/ed &ancy mechanisms toget into cells D these are a//lied &orgene trans&er. irus7constructs aregenerated* which contain the target gene"ut not genes &or ,irus re/lication genes

essential &or generating the ,iral /articlesare su//lied "y ac%aging CellsX3.

Adeno,iruses: transient >/ression

8etro,iruses: sta"le integration "uttarget !ust /roli&erating cells

$enti,iruses: sta"le integration* alsotransduce uiescent* non7/roli&eratingcells.



25

50

Adenovirus Retrovirus

•Episomal gene expression •Long-term, stable gene expression; inheritable

•Infects dividing &nondividing cells

•Infects dividing cells only

•High-level proteinexpression

•Moderate protein expression

•iral titers of !p to "#12

pf!$ml

•iral titers of !p to "#6 cf!$ml %an be

concentrated to "#9 cf!$ml'

•(ccommodates inserts of !pto ) *b

•(ccommodates inserts of !p to + *b

•Elicits imm!ne reactions in

vivo

•.oes not elicit imm!ne reactions in vivo

51

>am/le o& andadeno,iral system



26

Adeno7associated ,irus AA3

is a small ,irus which in&ects human cells

is not causing any o",ious disease

causes a ,ery mild immune res/onse

can in&ect "oth di,iding and uiescent cells

AA ,ectors /ersists mostly in an etrachromosomal statewithout integrating into the genome o& the host cell the nati,e,irus can integrate to some etent into the host genome3.

romising gene thera/y ,ectors clinical trials ha,e "een done&or C8* hemo/hilia B* arthritis ;3

52

53



27

54

ackaging cell line: %oenix cells. T%ese cells must not +e too confluent since t%is ,ill lo,er t%eproduction of )irus significantly.

"ay 1

late p%oenix cells on 1 cm plate &1.-1. x 1/ cells(

"ay 2

&&Transfect p%oenix cells using 2µ

g "#A and µ

l 0ugene 1.3 ml Opti4E4.'ncu+ate 5/ min !T and add to cells.(( O! +etter use CaO6777

"ay 5

Carefully remo)e old medium and add 2 ml of fres% medium.

'ncu+ate at 52°

C for 26% or 63% &or 2%(.

"ay 6-

8ar)est supernatant after 26-63%. 9irus sup can +e %ar)ested until t%e cells start looking un%ealt%y.

ut supernatant in / ml tu+e in ice +ucket in t%e %ood. Add 22 ml fres% medium to t%e packagingcells and put +ack into incu+ator.

Spin )iral sup to pellet any remaining cells.

0or storage of )irus: transfer to cryotu+e and snap freee ,it% #2 store at -3/°

C. ;pon freeing )irustiter goes do,n roug%ly t,ofold70or use straig%t a,ay: 0ilter )irus sup t%roug% a /.6-

µ

m cellulose acetate or polysulfonic filter&do #OT use nitrocellulose filter since it +inds proteins in t%e retro)iral mem+rane7(. <eep onice until use.

rotocol:roduction o& retro,irus "y trans&ection o& /ac%aging cells

55

#ay 4

S/lit the target cells into 10 cm dishes.

#ay 6

Add ? µgml /oly"rene to the &iltered ,iral su/ernatant. Mi gently "yin,ersion.

8e/lace the media in the target cells with the ,iral su/ G /oly"rene.Incu"ate at 4<°C &or h and add eual amount o& media containing/oly"rene.

#ay 5

8e/lace the media.

#ay

8e/lace the media with &resh media containing /yromycin to select &ortrans&ectants. he amount o& /yromycin used is determined "y %illing cur,ee/eriments. Select colonies o& sta"le trans&ectants %ee/ing the /yromycinin the media.

rotocol II:In&ection o& M>s with recom"inant retro,irus &or

ma%ing sta"le cell lines



28

56

8e/orter7(ene Assays

E=0 >nhanced (reen luorescent rotein3 and ,ariants thereo& e.g.desta"ilied >(3

$uciferase

CAT Chloram/henicol7Acetyl7rans&erase3

>-=alactosidase &lac?(

SEA secreted al%aline /hos/hatase3 etc.

>nymes or other molecules* which are easily detecta"le* area//lied as Hre/orter molecules to detect /romoter acti,ities or theacti,ities o& signaling /athways.

he re/orter gene is cloned into an a//ro/riate /lasmid e.g.mammalian e/ression ,ector3 and trans&ected into the cells o&interest* &ollowed "y the "iological e/eriment e.g. stimulation3.

>am/les &or re/orter7genes:

57

8egulated romoters in 8e/orter (ene Assaysarti&icial /romoters3

andem 8e/eats o& transcri/tion &actor "inding sites e.g. 5 97%a//aB3. -&ten commercially a,aila"le e.g. &rom Stratagene or Clontech3 can "e used to determine the acti,ation o& a certain transcri/tion&actor or signaling /athway3

8e/orter (eneminimal/romoterelement



29

58

8egulated* 9atural romoters in 8e/orter (ene

Assays

9atural /romoters usually contain "inding sites &or se,eraldi&&erent transcri/tion &actors sometimes se,eral co/ies o& asingle "inding site3 are regulated "y se,eral signaling/athways.>am/le:

IRF-1 GRE AP-1 NF/IL-6NF

κ

B

p65 / c-Rel

IL-8

Promoter

can "e used to determine the regulation o& a s/eci&ic /romoter o& interest$uci&erase is usually easier to measure with high sensiti,ity than the gene o&interest in this case I$7?3

$uci&erase

59

9ormaliation constructs with constituti,e /romoters

C49 : &rom human Cytomegalo7irus: induces strong constituti,ee/ression &ast* a"out 1 day a&ter trans&ection3

!S9 : &rom 8ous Sar%oma7irus: wea%er* "ut ,ery constant* constituti,ee/ression* slightly slower ta%es 2 d3

S96/: Simian irus 607romoter

Actin-romoter: human /romoter o& a Hhouse%ee/ing gene

;+i@uitin-romoter: human /romoter o& a Hhouse%ee/ing gene

;usually used &or the normaliation control in re/orter gene assaysto com/ensate &or di&&erences in trans&ection e&&iciency* etraction e&&iciencyor ,ia"ility o& cells in di&&erent sam/les3

+sual setting: athway7s/eci&ic re/orter construct e.g. with &ire&ly luci&erase3G constituti,e normaliation construct e.g. +"iuitin7romoter dri,en β7galactosidase or 8enilla7luci&erase3 uanti&ication o& normalied ,alues

$uci&erase β7galactosidase or ire&ly 8enilla7luci&erase3

romoters for t%e normaliation )ector:



30

>am/le o& an 8e/orter (ene Assay

60

46.36

0.90 0.491.94

26.66

4.63

0

5

10

15

20

25

30

35

40

45

50

p53 Rb E2F

f o l d o

f c o n

t r o l v e c

t o r

EP-cells

DU145

Acti,ities o& di&&erent signaling /athways or molecules /54* 8"* >23 areassessed with re/orter constructs containing res/ecti,e transcri/tion&actor "inding sites u/stream o& a luci&erase ,ector.

or normaliation /ur/oses a constituti,ely e/ressed control gene hasto "e cotrans&ected e.g. β7galactosidase downstream o& a constituti,e/romoter e.g. u"iuitin7/romoter3. alues are calculated as $uci&eraseβ7(al.

$ u c i & e r a s e β 7 ( a l

61

8e/orter7(ene Assay Systems &or Analyses o&Signaling athways

(ene o& interest

8e/orter7lasmid

ranscri/tion &actor construct

In case that the gene o& interestsacti,ates the s/eci&ic /athway*which leads to /hos/horylationand acti,ation o& the usedtranscri/tion&actor construct* thee/ression o& the re/orter e.g.luci&erase3 is induced.

ranscri/tion &actor construct

8e/orter7lasmid



31

62

#etection o& re/orter genes: ( and ,ariants3

luorescence measurement "y&luorometry e.g. in @7well&luorescence readers3

Microsco/y

low analysis cytometry*ACS: luorescence acti,atedcell sorting: (7containingcells can "e se/arated &romother cells and i& necessaryalso &urther culti,ated a&ter/uri&ication3

( &luorescence

63

#etection o& $uci&erase ire&ly or 8enilla $uci&erase3

PMTLuciferase-

Zellextrakt

ATP

Luciferin

M: &rom htt/:www.moleculare/ressions.com

ery sensiti,e detection in cell etracts "y measuring theluminescence generated &rom luci&erase in /resence o&luci&erin and A. he su"strate $uci&erin has to "e in!ectedinto the sam/le* and measured immediately e.g. "yintegrating &or 5 sec3 as the emitted luminescence decaysuic%ly. >mitted /hotons are measured with /hotomulti/liertu"es.Measuring de,ices: $uminometeralso as @well de,ices a,aila"le3



32

64

#etection o& β7(alactosidase lac)3

o&ten "y /hotometry e.g. in >$ISAreaders3 using a yellow su"strates* whichturns red in /resence o& (alactosidasesu"strate: C8(* Chloro/henolred7β7#7(alacto/yranoside3. #etection at 5@5 nm.

Alternati,e: Chemiluminescencemeasurement

Alternati,e: luorimetric #etection:Su"strate: e.g. 47car"oyum"elli&eryl"eta7#7galacto/yranoside* C+( iscon,erted to a &luorescent /roduct

Microsco/ical detection: with Y7(al 57Bromo767chloro747indolyl7Z7#7galactoside3as su"strate: /roduces a dar% "luereaction /roduct

65

$yse cellsrecommended cell lysis "u&&er: 0.25M risECl /E ?.0* 0.25P ,,3 960*2.5 mM >#A3

i/et a"out 10 Ql o& etract into a @7well /late a//ro/riate neg. control "lan%: 10 Ql o& moc%trans&ected cells* or non7trans&ected cells D as there is a slight endogenous "7(al acti,ity3 D lea,e onewell em/ty &or "lan% A713

Add 100 Ql su"strate solution to the wells also to the "lan%7well3

Incu"ate until red color de,elo/s min to hours D de/ending on "7(al acti,ity* i& you ha,e low acti,ityyou can also incu"ate at 4<°C3

-/tional: Sto/ with 50 Ql o& Sto/ solution only necessary i& you want to time it eactly* e.g. "y adding

the su"strate in a timed way and sto//ing the reaction in the same way3

Measure with >$ISA 8eader at 5<0 nm ilter [43 o/timum: 5@5 nm

$ysis Bu&&er:

0.25M risECl /E <.6 or "etter ?.03

0.25P ,,3 960

2.5 mM >#A

C8(7su"strate solution: 1 mgml \ 1.5 mM3

in BS G 10 mM KCl* G 1 mM MgCl2

alternati,e su"strate "u&&er:

0 mM 9a2E-6 /E ?.0* 1 mM MgCl2* 10 mM KCl* 50 mM Merca/to7ethanol

Sto/ solution: 0.5M 9a2C-4

β7(alactosidase Assay with C8(



33

66

Methods to su//ress gene e/ression89A inter&erence3

Antisense7echnologies: to su//ress gene e/ression were started many yearsago. >arly a//roaches used antisense7oligonucleotides D "ut the e&&ect was ,ery,aria"le.

Alternati,e a//roaches used long antisense7strands hy"ridiing to the m89A:his usually leads to downregulation o& gene e/ression D "ut uite o&ten notonly &or the targeted gene D "ut also uns/eci&ically &or other genes. he reasonis that this is Hsensed "y the cells li%e a long ,iral ds89A* leading to ,irusde&ense mechanisms: acti,ation o& K8 /rotein %inase 83* /hos/horylation o&translation &actors and general downregulation o& /rotein synthesis.

Some years ago* scientists &ound that small ds89A in the range o& 1@721nucleotides inter&eres s/eci&ically with target genes* without a&&ecting othergenes small inter&erent 89A* si89A3 D "ecause they are too small to acti,ate,irus de&ense mechanisms.

67

rinci/le o& 89A7inter&erence

Small ds89A si89A3 "inds to an89A7induced silencing com/le8ISC3F consisting o& argonaute/roteins.

ogether with the 8ISC* one 89Astrand "inds to the target m89Aand leads to s/eci&ic degradationo& this m89A.

he 8ISC com/le is Hrecycledleading to degradation o& additionm89A7targets.



34

micro789AOs D the "iological mechanism to

su//ress gene e/ression

mi89As D can ha,etwo e&&ects:

13 m89A degradation

23 inhi"ition o&translation thise&&ect o&ten doesnot reuire a

100P match withthe target m89AU3

69

Chemically synthesied si89A

htt/:www.dharmacon.com

htt/:www.mwg"iotech.comhtmls]synthetic]acidss]rna.shtml

he two short 89As are annealed and trans&ected usually usingmethods that are suited &or short oligonucleotides e.g.$i/o&ectamine2000 &rom In,itrogen* Ytreme(ene &rom 8oche;3.

#esign: traditionally the &irst AA7#u/lett is searched D and the &ollowing 1@"ases are chec%ed &or (C7content should "e 60 D 50P3* the seuenceshould "e target gene s/eci&ic chec%ed "y B$AS3 D an a//ro/riate 89Aseuence and the re,erse com/lementary 89A are chemically synthesiedand ordered "y a com/any* e.g. #harmacon* In,itrogen* ML(*;3.

Com/any home/ages o&ten o&&er a "asic si89A design:



35

70

ector7coded si89A small hair/in 89A sh89A3

the normal trans&ection methods* o/timied &or /lasmids can "e used

Anti"iotics selection genes e.g. (61?* /uromycin;3 can "e included sta"le H%noc%7down cell lines can "e generated www.imgene.com*htt/:www.oligoengine.com3

71

small hair/in 89A: hair/in7loo/



36

72

ro&essional #esign o& si89A or sh89A

#esign ,ia com/any we"sitehtt/:www.thermoscienti&ic"io.comdesign7center^redirect\truehis deli,ers a list o& se,eral /ossi"le seuences gene s/eci&ic*chec%ed "y B$AS3 D with a score "ased on em/irically determinedcriteria: 9ature Biotechnology 22* 427440* 2006

Chec% literature &or &unctional si89A seuences

or transduction o& /rimary cells: lenti,iral sh89A constructsalso wor% in non di,iding cells3

there are also induci"le lenti,iral constructs a,aila"lehtt/:tronola".e/&l.ch3

Many ,ectors can also "e o"tained &rom /lasmid re/ositories: Addgene: htt/:www.addgene.org

Belgian re/ository: htt/:"ccm."els/o."ed"lm"/]search]&orm./h/

(ene re/lacement strategy

73

gene o& interest5O+8 4O+8

si89A targeting the endogenous

m89A ,ia theuntranslated region

mutated gene o& interest

&oreign4O+8

S60olyA3

the mutated genere/laces the endogenousgene

good/romoter

>/ression /lasmid containing:

endogenous m89A



37

Im/ortant controls in si89A e/eriments

scram"led si89A as negati,e control

mutated si89A with some mismatch as negati,e control note:might act as mi89A U3

other si89As targeting the same m89A should ha,e the samee&&ect

i& you use sh89A ,ector "ased 89A7inter&erence3 use anunrelated sh89A as negati,e control e.g. sh89A ,ector withscram"led si89A seuence3.he em/ty sh89A ,ector is not a ,alid negati,e control

75

Micro-Injection:

This allows injecting antibodies

against certain endogenous

proteins > interfering with their

functions. However, just a limited

number of cells (e.g. up tohundreds with automated systems)

can be targeted > the following

analysis should be a single cell-

based assay, such as microscopy.

Alternati,e Methods to in&luence endogenous/rotein le,els



38

76

8esearch Methods 7 -,er,iew

cell culture systems


analyses o& cellular com/onents

analyses o& molecular interactions


microsco/y

&low analysis ACS3

analyses o& cellular /rocesses /roli&eration* a/o/tosis..3

77

Analysis o& roteins "y S#S7A(>

S7S

S-67

7

77

77 777

7 7 77

7777

77

reducing agent #3or Merca/to7>thanol7 "rea%s disul&ide

"onds@5°C

S#S D sodium dodecyl

sul&ate: coats /roteinswith negati,e charges



39

78

S#S7(els

For final concentration of gel ( % T):

Separating gel (10 ml)

Stack gel

(10 ml)

7% 10% 12,5 % 15% 5%

30% Acrylamide-bissolution 29:1(A) 2.33 3.33 4.17 5 1.67

4x Separation buffer

2.5 2.5 2.5 2.51.5 M Tris/HCl pH

8.8

4x Stacking buffer

2.5

0.5 M Tris/HCl pH6.8 + phenolred

aqua dest. 5 4 3.2 2.4 5.7

SDS (10 %) 0.1 0.1 0.1 0.1 0.1TEMED 0.015 0.015 0.015 0.015 0.015

APS (10%) 0.03 0.03 0.03 0.03 0.03

2 M Tris-Cl

(pH 6.8)

2.4 ml

SDS 0.96 g

Glycerol 4.8 ml

DTT 739 mg

Bromophenol

Blue4.8 mg

S#S7"u&&er

79

Coomassie7Blue staining: ro"ust*moderate sensiti,ity limit ≈ 1 Qg3

Sil,er staining: elementary sil,er isde/osited at the site o& /roteins*,ery sensiti,e limit ≈ 10 ng3

/rotein7s/eci&ic &luorescent dyes:S=8-7-range* S=8-78u"y*#ee/7ur/lecom/ati"le with MA$#I7-* MS3

S/ecial stainings: roteoglycansAlcian7Blau3* glyco/roteinsSchi&&Os 8eagent3

Autoradiogra/hie* luorogra/hy

#etection techniues



40

80

k"a

2 k"a

se/aration with 12P Acrylamide: 20 7 ?0 %#a

Sil,er staining

Molecular weight assessment a&ter S#S7A(>

81

htt/:www.meduniwien.ac.atuser!ohannes.schmidS#S7A(>.ls

logMLT

migration distance starting &romstac%ing gelse/aration gel inter&ace



41

82

Sensiti,e detection o& radioacti,ely la"eled /roteins

(el is euili"rated with a radiosensiti,e &luoro/hore:e.g. #i/henyloaole -3* Sodiumsalicylate

#etection "y Y7ray &ilm or hoshor7Imager de,ices

luorogra/hy* Autoradiogra/hy

83

hos/ho7Imager detection1 day e/osure3

luorogra/hy

Y7ray &ilm detection4 months e/osure UU3



42

84

Solutionsiing solution: 50 P ethanol* 10 P glacial acetic acid* ad 100 P with aua dest.Incu"ating solution 1$3: 40 P ethanol* sodiumthiosul&ate anhydrous 2g* sodiumacetat

anhydrous 46 g* &ill u/ to 1$ with aua dest. Be&ore use add 125 Q$ o&glutaraldehyde50 m$ incu"ating solution.

Sil,ernitrate solution 1$3: Ag9-4 1 g* dissol,ed in 1$ aua dest.. Be&ore use add 10 Q$o& &ormaldehyde50 m$ o& sil,er nitrate solution.

#e,elo/ing solution 1$3: 9a2C-4 anhydrous 25 g* dissol,ed in 1$ aua dest.. Be&oreuse add 10 Q$ o& &ormaldehyde50 m$ o& de,elo/ing solution.

Sto/ solution 1$3: sodium7>#A 15.<? g dissol,ed in 1$ aua dest..

A&ter electro/horesis* the /olyacrylamide gel is ta%en out o& the casting sandwich and/laced in a clean glass "ea%er &illed with &iing solution. All &ollowing ste/s arecarried out while gently sha%ing. he gel has to "e incu"ated with the &iingsolution &or 40 minutes. A&ter &iation an a//ro/riate amount o& incu"ating solutionincluding glutaraldehyde the gel has to "e at least co,ered "y liuid3 is /re/ared

and added to the gel* &ollowed "y incu"ation &or 15 minutes* discarding the &iingsolution and washing with aua dest. 4 &or 5 minutes and 10 minutes incu"ationin sil,ernitrate solution including &ormaldehyde. he sil,ernitrate solution iscollected s/ecial waste3. #e,elo/ing is carried out "y incu"ating the gel inde,elo/ing solution including &ormaldehyde until the desired intensity o& /roteinstaining is reached* &ollowed "y discarding o& de,elo/ing solution and adding sto/solution. he gel should incu"ate &or at least 1 hour in the sto/ solution. A&terwards the gel can "e stored in aua dest. or dried with ,acuum.

Sil,er Staining o& A(> (els

>MSAOs >lectro/horetic mo"ility shi&t assays3

;used to monitor acti,e transcri/tion &actors "y "inding to short* la"eledoligonucleotides com/rising the "ound #9A seuence3

>am/le: com/.: com/etitor: non7la"eledds7oligo o& the same seuenceusually added in 107&oldmolar ecess3 D com/etes withthe la"eled oligo &or "inding tothe reduces the s/eci&icsignal

mut.com/.: mutated com/etitor:should not com/ete &or s/eci&ic"inding



43

#e&ining the com/ositiono& 7com/lees using

anti"odies and su/ershi&ts

su/ershi&t

>MSA Alternati,e: ABC# AssayA,idin7Biotin Com/le with #9A3

87

Stre/ta,idin

Biotin

ds-ligo



44

88

Se/aration o& /roteinsaccording to their isolelectric/oint /E at which they arenot charged3

+sage o& immo"ilied /E7gradients Am/holines3

nati,e I> or denaturing I>urea3 can "e done

Isoelectrical ocussing I>3

89

&or analyses o& com/le /rotein mitures

→ roteomics3

com"ination o& I> 1. dimension3 and S#S7A(> 2.dimension3

high se/arating resolution > 1000 S/ots3 rotein /attern data"ases are a,aila"le and so&tware

&or /attern com/arison

ident&ication o& s/ots "y mass s/ectrometry or "yimmunological methods immuno"lotting3

2#7>lectro/horesis



45

90

rinci/le o& 2#7>lectro/horesis

91

2#7S#S7A(>



46

2#7#I(> #i&&erence (el >lectro/horesis3

rotein etracts &romtwo di&&erent sam/lesare la"eled with twodi&&erent &luorescentdyes e.g. Cy2 and Cy43and mied usuallytogether with a /ooledstandard miturela"eled with a 4rd dye*eg. Cy53.

2#7A(> is /er&ormed*&ollowed "y scanning o&the gel with the 4wa,elengths ecitingthe 4 dyes the resultsare com/ared "ycom/uter analysis

(el chromatogra/hy sie7eclusion chrom.3

Se/aration techniue that uses /orous "ead material in acolumn to se/arate macromolecules according to sie.

Lide range o& molecular weights that can "e se/arated

Much larger molecules can "e se/arated than with S#S7A(> ornati,e A(>

93



47

(el chromatogra/hy

94

larger molecules are eluted &irst

(el chromatogra/hy

95

0.001

0.003

0.005

0.007

0.009

0.011

0.013

0.015

0.017

0 5 1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

1 0 5

1 1 0

min

E x

4 3 6 / E m

5 1 0

-0.01

0.01

0.03

0.05

0.07

0.09

0.11

0.13

0.15

0.17

0.19

T r p - f l u

o r e s c e n c e

SERT

Standards

670 kDa

158 kDa

44 kDaSERT

>am/le



48

S/in desalting or "u&&er echange "ased on gelchromatogra/hy

1. lace s/in column in a 1.5 m$ microcentri&uge collection tu"e.

2. Centri&uge at 1500 g &or 1 minute to remo,e storage solution.

4. Add 400 _$ o& desired &inal "u&&er to the resin "ed and centri&uge at 1500 g &or 1 minuteFdiscard &low7through

6. lace the euili"rated s/in column into a new collection tu"e

5. $oad your /rotein sam/le 140 Ql3

. Centri&uge at 1500 g &or 2 minutes to collect desalted sam/le.

<. rotein sam/le is now desalted ` "u&&er echanged and ready &or use

96

97

Lestern Blotting: >lectro/horetic /rotein7trans&er&rom a gel mostly S#S7A(>3 onto a mem"rane

Mem"ranes: 9itrocellulose*oly,inyl7di&luorid #3

detection ,ia enyme7cou/led anti"odies

>nymes: 7 Eorse 8adish eroidase E83

7 al%aline /hos/hatase A3

Immuno"lotting



49

98

Immuno"lotting II

99

Let Blottingeui/ment



50

100

Blotting conditions

101

Semidry "lotting Bio8ad3

wet Milli/ore Immo"ilone7mem"rane in Me-E* rinse withwater* then 1 min. in Blotting "u&&er:25mM ris Base150 mM glycine10P methanolma%e a @0P stoc% without Me-E*

which is then added &reshly3&ilter 0.2 mm UUU wet 5 4MM &ilter /a/ers 7 anode then wetted mem"rane then gel then 5 wetted &ilter /a/ers wet cathode* close lid uic%ly

without mo,ing "ac% and &orth. "lot 250 mA &or a small Bio78ad gel

should "e 20* increasing to 40 3&or 40 min.



51

102

Chromogenic

detection o& E8

Immuno"lotting: ,arious su"strates

Chemiluminescence

7su"strate

103

Lestern Blot rotocol

"etection: "loc% 1 hour to -9 in BS5P mil% at 8 wash 4 BS0.1Pween 20 1st AB in BSween* 1 h wash 4 5 min BS0.1Pween 20

add 2nd AB in BSween e.g.* /eroidase7con!ugated don%eyanti7ra""it Amersham* at 1:10.0003 &or 1 hour

wash 4 5 min

EC$-system: com"ine eual ,olumes o& soln. A and soln. B Amersham >C$ or

ierce Su/erSignal Lestico3 add to &ilter* incu"ate 1 min. Amersham3* or 5 min ierce3 roll away* co,er with saran wra/* e/ose within 10 min. e/osure times can ,ary "etween seconds and 20 min.3



52

104

Ad,antages: almost no costF sta"lere/roduci"le* stored in &roen aliuots

o 10 mls o& 100 mM ris /E ?.5 83* add

50 Ql luminol warm to redissol,e3

22 Ql o& coumaric acid warm to redissol,e3

4 Ql o& E2-2 &resh months3

our onto "lot &or 1 minute and /rocess as normal 10 ml enough &or 100cm23

Stock luminol: 250 mM 47amino/thalhydraide lu%a [0@2543F 2mgs in mls #MS-F store &roen in 0 Ql aliuots.

Stock coumaric acid: @0 mM coumaric acid Sigma C@00?3: 4? mgs in2.5 mls #MS-F store &roen in 25 Ql aliuots.

ST!''#= of mem+ranes: with 2P S#S* 2.5mM 8IS /E.?*100mM Beta7merca/toethanol &or 40 min at 50°C

>C$ reagent: sel&made ,ersion

105

Interaction "etween anti"ody andantigen in solution

Isolation o& immuno7com/lees "yrotein A7 Agarose Se/harose3 D Beadsrotein A \ Ig(7"inding /rotein o& Sta/hylococcus aureus

Alternati,e: rotein (: o&ten used &ormouse monoclonal anti"odies* which are notwell "ound "y rotein A

7 or anti"ody co,alently lin%ed to acti,ated

Se/harose C9Br7acti,ated3

Analysis "y S#S7A(>

Immuno/reci/itation Antigen



53

106

>am/le o& an Immuno/reci/itation

roteins la"eled uns/eci&ically with 45S7methionine s/eci&ic detection o& a/rotein o& interest "y immuno/reci/itation &luorogra/hy

A&&inity chromatogra/hy

"eads can "e cou/ledwith anti"odies or othera&&inity ligands* which"ind a molecule o&interest.

a&ter washing o& the"eads* the ca/turedtarget molecules can "eeluted e.g. "y lower /E.

107



54

108

/roteolytic /rocessing

(lycosylation

hos/horylation

+"iuitination

;.

Analysis o& /ost7translationalmodi&ications

109

in ,i,o: culti,ation o& cells in /resence o& selecti,e/roteinase inhi"itors

inhi"ition o& intracellular /roteinase in cytosol* >8*

(olgi3 is /ossi"le !ust with mem"rane /ermea"leinhi"itors

inhi"itors that are not mem"rane /ermea"le act !ustin the etracellular en,ironment* in endosomes andlysosomes

in ,itro: Incu"ation with s/eci&ic /roteinases

Analysis o& /roteolytic /rocessing



55

110)7A7CE92 .... Benyloycar"onyl7henylalanyl7Alanyl7

#iaomethane inhi"its cysteine /roteinases3

)7A7CE92

roteolytic /rocessing in ,i,o3

111

in ,i,o: Biosynthesis in /resence o& inhi"itors

unicamycin: inhi"its the initiation o& 97(lycosylation

rocessing o& 97(lycans "loc%ed "y (lycosidase7Inhi"itors

#etection o& 97(lycosylation

unicamycin µgml3 0 0.1 0.5



56

112

>ndo E .... >ndoglucosaminidase EF clea,es !ustMannose7rich 97(lycans

9(ase ... e/tid:97(lycosidase V97(lycanaseV3

Analysis o& (lycosylation in ,itro3

113

in ,i,o: meta"olic la"elling with42Thos/hate e.g. in /resence ora"sence o& an e/ression or su//ressionconstruct &or a s/eci&ic %inase* &ollowed "yimmuno/reci/itation3

in ,itro:

7 Incu"ation with al%aline hos/hatase

7 hos/ho7aminoacid analysis #C3

7 Kinase7Assays

Immuno"lottingImmun/reci/itatione.g. with anti7/hos/ho7 s/eci&ic Anti"odyor &or instance anti7hos/hotyrosin3

Analysis o& hos/horylation



57

114

9(ase ... e/tid:97(ly%osidase V97(ly%anaseV3

Al%hos ... Al%aline hos/hatase

In ,itro hos/horylation Analysis

115

Immuno/reci/itated Kinase

anti"ody

Agarose7Bead

su"strate /hos/horylated su"strate

427γ A G

1. Immuno/reci/itation o& the %inase

2. Kinase78eaction in /resence o& 427γ 7A

4. S#S7A(> and luorogra/hy

in ,itro Kinase Assay



58

116

$ysis "u&&er &inal conc.3: &or 20 ml:

20 mM risECl /E<.5600 Ql 1 M150 mM 9aCl00 Ql 5 M25 mM "7glycero/hos/hate500 Ql 1 M2 mM >#A?0 Ql 0.5 M2 mM/yro/hos/hate600 Ql 0.1 M1 mM ortho,anadate200 Ql 0.1 M1P riton Y71002 ml 10P1 mM #20 Ql 1 M1 mM9a20 Ql 1 MA. dest.15.? ml

rotease Inhi"itors: added "e&ore use $eu/e/tin* e/statin* e&a7Bloc%3 according to stoc%

Kinase "u&&er &inal conc.3: &or 20 ml:20 mM risECl /E<.5600 Ql 1 M20 mM "7glycero/hos/hate600 Ql 1 M100 QM ortho,anadate20 Ql 0.1 M10 mMMgCl2200 Ql 1 M50 mM 9aCl200 Ql 5 M1 mM #20 Ql 1 M50 QM A50 Ql 20 mM1 mM 9a20 Ql 1 MA. dest.1?.<ml

$yse cells in wells3 with 500 Ql /er well o& $ysis "u&&er G /rotease inhi"itors3:20 min at 6°C.

Clear "y centri&ugation 16000 r/m* 6°C 15 min >//endor& centri&uge3. Sa,e an aliuot 40 Ql3 &or Lestern "lotting.

Immuno/reci/itate the %inase e.g. with 10 Ql anti7&lag a&&inity matri "eads* Sigma* &or &lag7tagged trans&ected %inaseF orwith a//ro/riate anti"ody &or endogenous %inase G rotein A7Se/harose or directly cou/led to agarose3: 2h at 6°Crotating3.

Lash the "eads: 4 with 1 ml BS 6°C3* 1 with 1 ml Kinase "u&&er 6°C3: /ellet the "eads "y centri&ugation 16000 r/m*

6°C* 65sec3 and remo,e the su/ernatant.re/are Kinase "u&&er: add MnCl2 to 10 mM stoc%: 1 M3 and 427g7A 5 QCi /er sam/le* usually 110 ,olume* i.e. 1 Ql

o& stoc% solution &or one 10 Ql assay3 and /reincu"ate at 40°C &or 10 min.

Add 1 Qg su"strate: (S7I%B 1 Ql3 or mutant su"strate as control3 to the "eadsF add 10 Ql %inase "u&&er* mi gently andincu"ate at 40°C &or 40 min or longer3.

Sto/ the reaction "y addition o& 6 S#S7sam/le "u&&er 6 Ql3 and /er&orm S#S7A(> with the sam/les* &ollowed "y &iationo& the gel 10P methanol* 10P EAc3* drying and autoradiogra/hy.

or detection with hast(el: use only 5 Ql "eads* 5 Ql %inase "u&&er* 0.5 Ql su"strate and 2 Ql 6 S#S7sam/le "u&&er: 8un a12.5P hast(el with 6 Ql /er sam/le

Kinase Assay7 rotocol

#etection o& +"iuitination

trans&ection o& a tagged u"iuitine.g. Eis7tagged3 together with thegene o& interest e.g. &lag7tagged3

immuno/reci/itation o& the geneo& interest

o/tional: resus/end and heat the

"eads in 1P S#S7"u&&er* dilute to0.05P S#S and re/eatimmuno/reci/itation to get rid o&/otential co7/reci/itating*interacting /roteins.

resus/end and heat the "eads inS#S7A(> "u&&er

S#S7A(>

Lestern Blot &or the Eis7tag

otential set7u/:



59

118

most o&ten used: density gradient centri&ugation

coarse se/aration: di&&erential centri&ugation

#etection o& su"cellular com/artments "y s/eci&ic

mar%ers enymes that are nearly eclusi,ely in thatcom/artment3

Su"cellular ractionation

Se/aration o& su"cellular com/artments3

119

#i&&erential centri&ugation

Su"seuent centri&ugation ste/s with increasing g7&orce



60

120

(radient centri&ugation:

Sam/les are usually layed on to/ o& a gradient* /roteinsor com/artments migrate through the gradient in Hones

121

(radients: continuous ↔ discontinuous Hste/sV3

sel&7&orming gradients e.g. ercoll3

density euili"rium centri&ugation3

sam/les are either layered on to/ o& the gradient or at the

"ottom

ractionation a&ter the centri&ugation e.g. "y /eristatic/um/ and &raction collector3

#ensity gradient centri&ugation

A B

A

B0

1000

100

,olume or time3Magnetic stirrer

gradient mixer:



61

122

Sucrose: low molecular weight 462 #a3*

osmotically acti,e

icoll: co/olymerisate o& Sucrose and>/ichlorhydrineF Mr ≈ 600 000 #a

ercoll: colloidal silica gel

s/ecial case &or #9A: Cesiumchloride

Su"stances to generate density gradients

123

Sel&7&orming gradients e.g. ercoll..3



62

124

Se/aration o& lysosomes 3 and (olgi 3

in a continuous ercoll gradient density b3

>am/le &or a density gradient centri&ugation

125





analyses o& molecular interactions &luorescence measurements

microsco/y

&low analysis ACS3




63

126

Methods to in,estigate macromolecular interactions

Interaction screening with /hages hage #is/lay3

=east 17Ey"rid System /rotein : #9A3

=east 27Ey"rid System /rotein : /rotein3

Mammalian 27Ey"rid System

(el7Chromatogra/hy

Co7Immuno/reci/itation

luorescence 8esonance >nergy rans&er 8>3 D see &luorescence methods

127

Interaction screening with /hageshage #is/lay3

A gene li"rary is e/ressed on thesur&ace o& a//ro/riate /hages e.g.M143* which are incu"ated with s/eci&ictarget /roteins immo"ilied on /lates.+n"ound /hages are washed o&&F "ound

/hages are eluted "y lowering the /E.3-4 x

Bound /hages are am/li&ied and againincu"ated with /lates containing the target/roteins D this re/eated 4 D 6 to enrich thes/eci&ically "inding /hages. Clones are isolatedand seuenced Seuence o& the "inding

/rotein



64

128

=east 17Ey"rid System

#9A78egion with /otential /rotein"inding sites in re/eats3

AA EIS lac)

(al67Acti,ation domain

Insert &rom li"rary

ranscri/tion

or the identi&ication o& /roteins that "ind s/eci&ically to a gi,en #9A seuence e.g.transcri/tion &actorsF #9A:/rotein interaction3.

he #9A seuence o& interest e.g. &rom a /romoter3 is usually cloned in re/eats 4753in &ront o& an a//ro/riate selection gene e.g. a histidine synthesis gene3 and ana//ro/riate re/orter yeast strain which is not ca/a"le o& growing in the a"sence o&histine3 is sta"ly trans&ormed with this construct. Su"seuently* this yeast strain istrans&ormed with a li"rary containing /utati,e "inding /roteins o&ten &used to thetransacti,ation domain o& the (al6 transcri/tion &actor3. Binding o& a /rotein to the #9Aseuence results in growth o& this yeast clone on selection /lates.

growth onselection /lates

129

=east 27Ey"rid System

A yeast strain is used* which does not contain a &unctional (al6 transcri/tion &actor D"ut re/orter and selection genes* which are downstream o& (al67de/endent /romotersEistidine7 and Adenine7synthesis genes* lac) &or β7(alactosidase e/ression* whichcan "e used &or staining3. his strain is trans&ormed with /utati,e interaction /artners:

In case o& an interaction "etween /rotein Yand = au&tritt* a &unctional transcri/tion &actor is"uild* which "inds to (al6 /romoters D and thecells can grow on selection /lates without Eisor Ade3.

2. &usion /rotein o& the (al67transacti,ation domain and rotein = or ali"rary insertF \ H/rey3

1. &usion /rotein o& the (al67#9A7"indingdomain and rotein Y H"ait3

Co7rans&ormation or com"ination "yyeast mating



65

130

re/aration o& a =east 27Ey"rid Screen

1. Cloning o& the gene o& interest into the "ait,ector in &rame with the (al67#9A7"inding domain3: selection in "acteria e.g.,ia Kanamycin3* selection in yeast e.g. ,iar/7synthesis gene33

2. est &or Auto7Acti,ation with Hem/ty(al6A#7,ector3: ests whether the gene o&interest interacts with the (al6 acti,ationdomain without the need o& /rotein =3:I& it does so* the "ait cannot "e used in theyeast 27hy"rid system.

he (al6 A# ,ector contains a second

selection gene &or yeast e.g. leucinesynthesis gene3. Co7trans&ormants o& thetwo ,ectors grow in the a"sence o& r/ and$euF "ut they should not grow in thea"sence o& the amino acid that issynthesied !ust when an interaction occurse.g. in the a"sence o& histidine oradenine3.

131

Com"ination o& the two /utati,e interaction /artners

X

PCR from single colonies (with primers specific for the library vector)

sequencing of the putativeinteraction partners

purification of PCR-Products

His, Ade,lacZ

Reporter strain Y187pretransformed with a library (inGal4AD-Vector), Mating Type: α

Reporter strain(e.g. AH109)

Mating Type: a

Mating: Incubation of the two haploidstrains for 24 h at 30°C, 40 rpm

> formation of diploid clones with bothvectorsclones, which contain interactionpartners grow on selection plates andexpress lacZ

Gal4ADX

Gal4BD

bait

Instead of mating the two vectors can becombined by classical transformation



66

132

>am/le o& a yeast two7hy"rid result

A3 single colonies on selection/lates S#7$eu7r/7Ade3

B3 Strea%ing out the colonies &rom the&irst selection /lates to secondaryselection /lates e.g. with higherselection /ressure and stringency:S#7$eu7r/7Ade7Eis3

133

eri&ication o& a yeast 27hy"rid result

1. Analysis o& the seuence and com/arison with data"ase: chec% whether the -8is -K in &rame with the (al6A#3

2. Isolation o& lasmid7#9A &rom the yeast colony

4. 8e7trans&ormation in >.coli to se/arate "ait and /rey D using di&&erent anti"ioticsresistance3 and /re/aration o& the /lasmid containing the li"rary insert

6. Halse ositi,e est in yeast: rans&ormation o& the (al6A#7/lasmid containingthe identi&ied H/rey with the em/ty (al67"inding domain ,ector: this should notlead to growth on selection /lates o& interaction i& there is growth* then theli"rary insert interacts with the (al6B# and not the "ait /rotein3

5. β7(alactosidase7assays also uantitati,e* to com/are interaction /artners3

. eri&ication in the correct cells human cells3* e.g. "y co7immuno/reci/itation



67

134

>am/le o& a alse7ositi,e estX

>am/le o& lac)uanti&ication

1

1,1

1,2

1,3

1,4

1,5

1,6

1,7

neg. control IKK2/ GMRa IKK2/GMRb

r e l . a c

t i v i t y

135

Solutions: Synthetic dro/ out solution 10 in A#: $7isoleucine 400 mg$* $7,aline 1.5 g$* $7adenine hemisul&ate salt 200

mg$* $7arginine ECl 200 mg$* $7histidin ECl monohydrate 200 mg$* $7leucine 1 g$* $7lysine ECl 400 mg$* $7methionine 200 mg$* $7/henylalanine 500 mg$* $7threonine 2 g$* $7try/to/han 200* $7tyrosine 400 mg$* $7uracil 200 mg$

S# 7r/ medium synthetic dro/out medium3: synthetic minimal medium lac%ing try/to/han: yeast nitrogen"ase without amino acids .< g$* 2 P detrose glucose3 sterile detrose solution is added a&ter autocla,ingto a,oid maillard reactions3* /E ad!usted to 5.?* &or /lates : agar 1.5 g$

=# yeast /e/tone detrose3 "roth* yeast com/lete medium: yeast etract 10 g$* /e/tone 20 g$* 2 Pdetrose glucose3* /E ad!usted to 5.?

Aua dest. sterile $iAc 100 mM sterile $iAc 1 M sterile Bacterial 89A* used as carrier oly7ethyleneglycol >( 50 P w,3 sterile &iltered

10 m$ o& S# 7r/ medium are inoculated with the a//ro/riate yeast strain and incu"ated at 40°C while sha%ing at 200r/m on. -n the net day -# at 00 nm is measured and the yeast culture is diluted with =# to -#00 0.1. Atotal ,olume o& 50 m$ diluted yeast culture is used &or &urther incu"ation. >,ery hour -#00 is measured until-#00 0.6 is reached 4 7 5 hours3. hen the cell num"er is calculated with a homa cham"er. 210< cellsm$are su&&icient &or 10 trans&ormations. he yeast is then har,ested "y centri&ugation at 4000 r/m &or 5 minutes*the su/ernatant is care&ully remo,ed and collected &or autocla,ing. he /ellet is resus/ended in 25 m$ sterile A# and again centri&uged at 4000 r/m &or 5 minutes. A&ter remo,ing o& the su/ernatant the /ellet isresus/ended in 1 m$ $iAc 100 mM. >cess o& $iAc is remo,ed "y s/inning the tu"es &or 15 seconds at &ull s/eedin a ta"leto/ centri&uge and care&ully remo,ing the su/ernatant. he yeast /ellet is "rought to a &inal ,olume o&500 Q$ with $iAc 100 mM. Aliuots o& 50 Q$ are /re/ared. -ne 50 Q$ aliuot o& this yeast sus/ension is used &orone trans&ormation. 50 Q$ aliuots are again "rie&ly centri&uged to /ellet the cells* the su/ernatant is remo,edand on to/ o& the yeast /ellet* layers o& the &ollowing trans&ormation solutions are /i/etted in &ollowing order:260 Q$ 50 P >(* 4 Q$ $iAc 1 M* 4.4 Q$ o& "acterial 89A 41 QgQ$3* <0.< Q$ sterile A#* 1 Qg / lasmid #9A1QgQ$3. he tu"e is then thoroughly mied "y ,orteing &or 1 minute until the yeast /ellet is com/letelydissol,ed and /laced &or 40 minutes in a 40°C water "ath. he tu"e is then trans&erred to a 62°C water "ath &or25 minutes in order to /er&orm the heatshoc%. he trans&ormation mi is then "rie&ly centri&uged &or 15 secondsat 6 000 g < 000 r/m3 in a ta"le to/ centri&uge* the su/ernatant is discarded and the /ellet is resus/ended in1 m$ sterile A#. 50 Q$ o& this trans&ormed yeast sus/ension are /lated on S# D $eu* 7 r/* 7 Ade /lates andincu"ated at 40°C &or some days.

#9A G water G carrier7#9A or 89A 1M $iAc

olyethylenglycol

yeast /ellet

$iAc =east rans&ormation



68

136

Mammalian 27Ey"rid System

Limitations:

1. not suited for screeningpurposes

2. Proteins are in the nucleus

and thus eventually not attheir normal localization

Posttranslational modifications such asphosphorylations, which might beessential for interactions are often notcarried out in yeast. In this case asimilar assay can be set up inmammalian cells (e.g. providing thekinase)

> FRET-Microscopy: as alternative tovisualize protein-protein-interaktion in

their physiological context

137

>am/le &or a mammalian 27Ey"rid est



69

138

Biochemical eri&ication o& rotein7Interactions

"y Co7Immuno/reci/itation CoI31. The 2 proteins of interest are transfected into mammalian cells (usually containing

to different tags, e.g. HA- and flag). 1 – 2 d after transfection, the cells are lysedand one protein is immunoprecipitated using antibody-beads against tag1 (e.g.flag). The beads are washed extensively with buffer (isotonic or hypertonic, nothypotonic) and finally heated with SDS-buffer to release bound proteins. SDS-PAGE and Western blotting is performed – using antibodies against tag1 andagainst tag2. If protein with tag2 co-precipitated with protein containing tag1, thenthere is interaction.

2. Co-immunoprecipitation of endogenous proteins (without transfection) – using thesame principle and antibodies against the endogenous proteins

Protein

X-myx

Protein

Y-HA1. 2.

bead

Antikörper

3.

HRP

139

>am/le &or a Co7Immuno/reci/itation

1. Co-IP with overexpressed proteins(after transfection); control:transfection with just one protein

2. Co-IP with endogenous proteinscontrol: IP with unrelated antibody



70

140

he salt concentration has an in&luence on the

stringency o& the co7immuno/reci/itationCo7Immuno/reci/itation o& 8A1 undIKK2 wor%s at 250 mM 9aCl* "ut not at

500 mM 8A18A2 interaction isstronger than 8A1IKK2 interaction

Co7Immuno/reci/itation o& 8A1 and8A2 at 500 mM 9aCl

141

Co7Immuno/reci/itation &or the #etection o& rotein Interactions

1. Transfection of cells with tagged proteins (one 6-well of CHO or HeLa cells is sufficient for one sample).

2. Preparation of extracts:

2.1. 1 d after transfection: wash cells with PBS

2.2. Lysis with 500 µl/well Lysis-Buffer + Protease Inhibitors: 15 min at 4°C.

Buffer: 0.5% NP40, 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 150 mM NaCl.

Protease Inhibitors: 10 µg/ml Aprotinin, 20 µg/ml Phosphoramidon, 40 µg/ml Pefabloc, 1 µg/ml Leupeptin, 1 µg/ml

Pepstatin (from 1000x stock solutions, Boehringer Protease Inhibitor set).The lysis is suited for cytosolic proteins and

membrane proteins. Nuclei remain intact (you can leave the nuclei on the plate when you take off the supernatant).

2.3. Spin the extracts for 15 – 30 min at 14 krpm, 4°C (HeLas: 15 min, CHO: 30 min)

2.4. Keep the supernatant and adjust the NaCl-concentration (150 mM – 1000 mM depending on the strength of

interaction; start in the range of 150 – 250 mM, increase the concentration if you want to increase the stringency)

3. Co-Immunoprecipitation3.1. Take 400 µl of extract for IP (keep about 30 µl extract for direct western analysis).

Use flat-top tubes (the visibility of the pellet is better in these tubes) Add 400 µl Lysis-Buffer/250 mM NaCl (without

NP40 > final concentration: 0.25%). Add beads (15 µl anti-flag-M2-Agarose, Sigma A-1205; alternatives: other

antibodies directly coupled to CNBr-activated Sepharose; Protein A- or Protein G-Agarose: the later will give more

unspecific binding). Rotate extracts + beads for 2 h at 4°C.

3.2. Spin for 30 sec at 14 krpm 4°C. Take off the supernatant, add 1 ml of lysis buffer/250 mM NaCl/without NP40

and invert tubes several times (do not vortex). Repeat this washing step.

3.3. Suspend the beads in 1 ml cold PBS and transfer the suspension to a new tube.

Spin 30 sec at 14 krpm, 4°C, take off the supernatant and repeat this washing step. Final centrifugation: 1 min at 14

krpm, 4°C. Remove the supernatant and suspend the beads in SDS-PAGE buffer (30 µl). Incubate for 5 min at 95°C

and pellet the beads for 2 min at 14 krpm.

4. SDS-PAGE

5. Western Blot: if possible use HRP-conjugated primary antibodies (anti-HA-HRP from Boehringer, anti-myc-

HRP from Invitrogen). This gives much lower background of unspecific bands (Ig light chain …).



71

ar Lestern Blotting

; the mem"rane is /ro"ed with a /rotein* which can "ind the /rotein o&interest. Lhile western "lotting detects certain /roteins using anti"odies* &ar7western "lotting detects /rotein:/rotein interactions.

143






microsco/y

&low analysis ACS3




72

144

rinci/le o& luorescence

1. electrons o& a &luoro/hore areecited "y a"sor/tion o& ana//ro/riate /hoton h ν −>3 andtheir energy state is raised to S1´

2. the ecitated state S1´eists &ora"out 1 D 10 nsec. >nergy is lost"y se,eral reactionsinteraction;3 leading to theecited state S1.

4. >lectrons &all "ac% &rom S1 to S0 D the energy di&&erence isreleased "y emission o& a /hoton which has lower energy than theecitation /hoton D and thus a longer wa,elength3 according toλ \ c ν

Ja"lons%i7(ra/h

rinci/les o& luorescence

dou"le "onds \ &lei"le delocalied3 /7electron system

&rom:

htt/:www.in,itrogen.comsiteusenhomesu//ortutorials.html



73

delocalied /7electron systems

alternating dou"le "onds3 caneasily a"sor" /hotons andthere"y "e raised to higherenergy le,els

delocalied /7electron systems

alternating dou"le "onds3 caneasily a"sor" /hotons andthere"y "e raised to higherenergy le,els



74

energy loss due tomo,ements* rotations etc...

... sudden &all &rom an ecitedenergy le,el to the groundstate



75

he a"sor"ance o& light/hotons3 de/ends on thecolour the wa,elength3

num"er o&a"sor"ed/hotons

ecitation s/ectrum



76

he emitted light /hotons3ehi"its a certain wa,elengths/ectrum colour3 Dde/ending on the nature o&the &luoro/hore

num"er and colour o&emitted /hotons

emission s/ectrum

153



77

154

>citation and >mission S/ectra

Stokes S%ift

In&os: htt/:www./ro"es.comser,letss/ectra

Ja,a7A//let &rom B#: htt/:www."d"iosciences.coms/ectra

155

Ja,a7A//let &rom B#:htt/:www."d"iosciences.coms/ectra



78

156

Characteristics o& &luorescent dyes

>citation Maimum: wa,elength o& maimal /hotona"sor"ance λ in nm3

>mission Maimum: wa,elength o& maimal /hotonemission &luorescence* λ in nm3

Molar >tinction coe&&icient: gi,es the a"sor"ance o&ecitation /hotons at the ecitation maimum λ in cm7

1M713

Wuantum7=ield: num"er o& emitted /hotons /er

num"er o& a"sor"ed /hotons.

Brightness \ molar etinction coe&&. uantum yield

157

arameters o& some im/ortant &luorescent dyes

dye > >m

#AI 45@ 61

IC 6@6 520

M8ho 550 5<4

eas8ed 5@5 15

#AI... 4’,6-Diamidino-2-Phenylindol

IC... luorescein Isothiocyanat

M8ho...etramethylrhodamine

8IC: etramethylrhodamine Isothiocyanate3



79

158

Alea7luoro/hores &rom Molecular ro"esIn,itrogen

www.in,itrogen.com3

159

( (reen luorescent rotein3 and its ,ariants

• Structure: barrel like with the chromophore in

the middle

• MW: appox. 29 kDa

• original protein from jellyfish (Aequorea

victoria), exists in bacterial and mammalian

codon optimized versions.

• Point mutations were incorporated improvingthe fluorescence (enhanced GFP: EGFP) and

also leading to other spectral variants (colours;

ECFP, EYFP…)

• Fluorescent in living cells – can be expressed

as fusion protein with the protein of interest (is

usually not altering the function of the target

protein)



80

160

luorescence ro/erties o& some (7ariants

ariant >citation nm3 >mission nm3

>B Blue3 4?0 660

>C Cyan3 644 6<5

>( (reen3 6?? 50<

>= =ellow3 514 52<

#s8ed 55? 5?4

161

-ther &luorescent /roteins

fluor. protein Ex-Peak nm

Em-Peak nm

quantum yield comment

EBFP 380 440 0.18 Clontech

ECFP 433 (453) 475 (501) 0.4 Clontech

EGFP 488 507 0.6 Clontech

wildtype GFP 397 (475) 509 0.77 Aequorea victoria

EYFP 513 527 0.61 Clontech

Citrine 516 529 0.76 Griesbeck et al. 2001

DsRed 558 583 0.29 Clontech, tetramer

DsRed2 563 582

0.55 tetramer,

HcRed1 588 618 0.02 Clontech, dimer

PA-GFP (Patterson

2002)

400 before act.504 (397)

after

515 before act.517 after act.

0.13 0.79

photoactivatable GFP, T203Hmutant of mammalian

codon-optimized wildtype

GFP

PS-CFP 400 before act.

490 after act.

468 before act.

511 after act.

0.2

0.23

photoswitchable CFP, turns from

cyan to green after intense

illum. at 405 nm

mOrange 548 562 0.69 Shaner et al., 2004

mStrawberry 574 596 0.29 Shaner et al., 2004

mCherry 587 610 0.22 Shaner et al., 2004

dTomato 554 581 0.69 Shaner et al., 2004, dimeric



81

hotocon,erti"le &luorescent /roteins

0

20

40

60

80

100

120

0 20 40 60 80 100 120

r e l f l u o r

sec

mOrange conversion to far-red(2x bleaching with 100% 488 nm in between)

mOrange

converted far red

control cell

m-range

ar78ed

hoto7switcha"le &luor. /rotein #ron/a

0

20

40

60

80

100

120

-20 0 20 40 60 80 100 120 140 160 180 200

% o

f i

n i t i a l f l u o r

sec

start bleaching at 488 nm

start reactivation

at 350 nm



82

164

luorimetric Analysis Methods

luorescence measurements are usually more sensiti,e than/hotometric measurements.Scanning &luorometers ha,e usually 2 monochromators &or ad!ustingecitation and emission wa,elengths. Most instruments also allow toad!ust the "andwidth o& ecitation and emission "etween 1D20 nm3he emitted &luorescence is measured "y a /hotomulti/lier tu"eM3 detector. he sensiti,ity o& that can "e ad!usted "y changingthe ,oltage e.g. 600 7 <00 3.

>mission

Monochromators#etector

165

arameters o& &luorometry

>citation wa,elength in nm

"andwidth o& the ecitation light 1 D 20 nm* Hslit width3

>mission wa,elength in nm

"andwidth o& the emission

sensiti,ity o& the detector Hgain* ,oltage o& the M3

Integration time o& the measurement slow D &ast* in sec.:in&luences the Hnoise3



83

166

La,elength Scans

0

0.5

1

1.5

2

460 480 500 520 540 560 580

nm

r e l . f l u o r .

>mission s/ectrum >C3

he eact emission andecitation /ea%s might di&&erslightly "etween di&&erent&luorometers.

or chec%ing the /arameters:7 run an ecitation wa,elengthscan at the literature ,alue o& the/ea% emission7 run an emission scan at thedetermined ecitation /ea% 7 re/eat the ecitation scan at

the determined emission /ea% or ad!usting these /arametersyou ha,e to consider the&luorescence /ro/erties e.g. theSto%eOs shi&t3 D to /re,ent thatecitation light is detected

ric%s &or o/timiing &luorescencemeasurements "ased on the s/ectra

nm

luor.

theor.ecitationcur,e

theor.emissioncur,e

real

emissioncur,e 1

ecitationwindow

/ossi"le emissionwindow

detected &luorescence

s/ill7o,er o& the ecitation light



84

ric%s &or o/timiing &luorescence

measurements "ased on the s/ectra

nm

luor.

theor.ecitationcur,e

ecitationwindowmorenarrow

emission cur,e 2


emission window

ric%s &or o/timiing &luorescencemeasurements "ased on the s/ectra

nm

luor.

real

emissioncur,e 1

real emission cur,e 4

le&t shi&ted"roader ecitation

window




85

170

ime Scans

0.001

0.003

0.005

0.007

0.009

0.011

0.013

0.015

0.017

0 5 1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

1 0 5

1 1 0

min

E

x 4 3 6 / E m

5 1 0

-0.01

0.01

0.03

0.05

0.07

0.09

0.11

0.13

0.15

0.17

0.19

T r p - f l u

o r e s c e n c e

SERT

Standards

670 kDa

158 kDa

44 kDaSERT

Can "e a//lied to determine the time course o& &luorescence changes e.g. todetermine enyme reaction %inetics D or &or instance in chromatogra/hy tomeasure the %inetics o& elution and thus the molecular weight o& a &luorescentcom/ound* such as a (7&usion /rotein3

171

ast Kinetic7AnalysisSto//ed7low luorometry3

wo reaction /artners are in!ected into a miing cham"er* where they are miedwithin a//ro. 1 msec "y sto//ing the &low.I& the reaction "etween the two com/ounds changes the &luorescence* this changecan "e recorded with a resolution in the microsecond range.

A

B

>citation light

luorescenceSto//ed low luorometry3

$ight7A"sor"anceSto//ed low hotometry3



86

172

>am/le &or a sto//ed7&low &luorometry

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0 0.02 0.04 0.06

seconds

r e l . f l u o r e s c e n c e

Tet-DNA

mutant T et-DNA

y = 734,21x + 244,09

R2 = 0,9508

0

100

200

300

400

500

600

700

800

900

0 0,2 0,4 0,6 0,8

DNA (µM)

r a t e ( 1 / s e c )

rate (15°C)

rate (37°C)

angent o& the initial &luorescence change reaction %inetics rate in 1sec3

$inear correlation "etween the initialreaction %inetics range and theconcentration o& the reaction/artners

K a&& \ 1K diss \ % on % o&&

% o&&

% on

% o&& % on

173

Wuantitati,e luorometry

1. >nyme7Measurements e.g. β7(alactosidase3

2. #9A7Measurements Eoechst 4425?* S=B8 (reen3: &luorescentdyes* which intercalate into the #9A and are &luorescentde/endent on the amount o& #9A

4. rotein7Measurements inherent &luorescence due to aromaticamino acids such as ry/to/hane dye: S=B8 -range* ;3

luorescence measurements can "e used to uanti&y a great ,ariety o&di&&erent su"stances.+sually a standard cur,e is measured with the o/timied measurement/arameters e.g. a&ter de&ining them "y wa,elength scans: ecitationand emission wa,elengths and corres/onding "andwidthsF M ,oltageand integration time3.

Some eam/les &or uantitati,e &luorometry



87

174

>am/le &or uantitati,e &luorimetricmeasurement

y = 0.8738x + 0.1417

R2 = 0.9959

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7 8

µg/ml

F l u o r e s c e n c e

( E m

3 7 6 / E x

2 7 6 ) ro+e

Standard cur,e

175

Con,entional C8:

Com/arison o& a gene o& interest with a house%ee/inggene using an end/ointdetermination

Sam/le 1 2

In real: sam/les contain di&&erentamounts o& c#9A* "ut this di&&erenceis not detected* when they reach thesame /lateau at the end o& thereaction this can "e re,ealed "ymeasuring the reaction /roduct "y&luorescence3 a&ter each cycle

1

2end/oint

luorescence as measurement ,alue in s/ecialanalysis techniues: 8eal7ime C8



88

176

C8 rinci/le

177



89

178

179

$ightCyclerM* 8oche

ca/illary withthe C87mi

heating

entilator

light source

dichroic mirrors

detectors

threshold

Ct

Scheme o& a C8 machine



90

8ealtime C8 machines

180

A//lied Biosystems

Ste/-ne lus8oche $ight Cycler

ca/illaries

a//. 1 Rsam/le3

@7well /lates

181

8eal7ime C8 with S=B8 (reen as #9A7&luorescence dye

S=B8 (reen intercalates in theam/li&ied ds#9A C87/roduct37leading to an increase in &luorescencewith increasing cycle num"er.

A&ter many cycles the &luorescencealso increases in the water control Ddue to the &ormation o& /rimer

aggregates



91

182

Melting /oint analysis o& the C87/roduct

S/eci&ic and uns/eci&ic C8/roducts can "e distinguished"y their di&&erent meltingtem/erature as determineda&ter the C8 "y slow heatingand the decrease o& the&luorescence at the melting/oint3. his can also "e a//liedto detect mutations.

measurement o& the &luor. a&ter each cycle at atem/erature a"o,e the melting tem/erature o& theuns/eci&ic C8 /roduct allows uanti&ying !ust thes/eci&ic /roduct

183

8eal7ime C8 with 8>7Ey"ridisation ro"es

Lithin the seuence &lan%ed "y rimer 1 andrimer 2 am/li&ication /rimers3* two additionaloligonucleotides Ey"ridisation ro"es 1 and 23are situated* which contain two di&&erent&luoro/hores at the 4O and 5O ends. Lhen theseoligos "ind to the C8 /roduct* the &luoro/horescome into close /roimity and one &luoro/horecan trans&er /art o& its &luorescence energy to theother one &luorescence resonance energytrans&er* 8>3* which then starts to shine. In

this case /rimer aggregates do not generate a&luorescence signal.



92

184

8eal7ime C8 with aMan /ro"es

A aMan7/ro"e contains 8>7#onor and Acce/tor Wuencher37luoro/hore within thesame oligonucleotide. At the annealing tem/erature o& the oligo* the /ro"e "inds to the C8/roduct. he eonuclease acti,ity o& the a7olymerase clea,es the /ro"e and leads toincrease o& the #onor7&luorescence due to de7uenching.

8ealtime C8 D Wuanti&ication o& geneu/down7regulation

y = -1.67ln(x) + 25.767R² = 0.9972

15

17

1921

23

25

27

29

31

33

35

0.1 1 10 100

C P

ng cDNA input

PCR efficiency

crossing point CP

Log. (crossing pointCP)

1. #etermine the C8 e&&iciency o& yourgene o& interest and that o& yourhouse%ee/ing re&erence3 gene usingserial dilutions e.g. o& /lasmids orc#9A /re/arations3: > \ ideally 2du/lication at each cycle3 "ut

realistically lower e.g. 1.?3

2. Calculate u/7 or downregulation o& your s/eci&ic gene o& interest usingthe di&&erences in the crossing /ointC3 ,alues with the euation:

>cel tem/late on my we"site3)(

)(

arg

)(

)( arg

samplecontrolCP

ref

samplecontrolCP

et t

ref

et t

E

E ratio

−∆

−∆

=

Ct7method

&a&&l ML: A new mathematical model &orrelati,e uanti&ication in real7time 87C8.9ucl Acids 8es 2001* 2@@3:e65



93

Calculating the C8

e&&iciency &rom thesha/e o& the cur,e

186

10

100

1000

10000

100000

0 10 20 40 60 50

l o g &

0 l u o r (

cycle

710000

0

10000

20000

40000

60000

50000

0000

<0000

?0000

0 10 20 40 60 50

0 l u o r

cycle

7 Lithout using a dilution cur,e

7 Can "e calculated &or eachsam/le se/arately

>cel tem/late on my we"site3

9eurosci $ett. 2004 Mar 14F44@13:27.

Assum/tion7&ree analysis o& uantitati,ereal7time /olymerase chain reaction C83data.

8ama%ers C* 8ui!ter JM* #e/re 8E*Moorman A.

$in8egC8 So&tware

187

htt/:www.hart&aalcentrum.nlinde./h/^main\&iles`su"\$in8egC8



94

188

189






microsco/y

&low analysis ACS3




95

Microsco/y: Euman ,ision and the conce/t o&

magni&ication

190

image &ormation in the human eye

27ste/ magni&ication /rinci/le o& amicrosco/e with 2 lenses:

o"!ecti,e and eye /iece occular3

191

Basics o& o/tical resolution I

ine structures induce a di&&raction o& light lighto& ero7order* 1st order ...3. $ight di&&raction on asmall iris is more or less eual to di&&raction onsmall cellular structures:

sinθ

(1)

1.22(λ

/d)

θ ... angle to the &irst light minimumλ... wa,elengthd ... diameter o& the iris

for very small angles θ: θ

(1)

1.22(λ

/d)o"!ects that are closer than

θ

(1) cannot "e resol,ed asse/arate o"!ects



96

192

Basics o& o/tical resolution II

he more orders o& light are resol,ed the "etter is the resolution.

he o/tical resolution that can "e achie,ed is de&ined "y the so callednumerical A/erture 9.A.3 o& the o"!ecti,e.

#.A. B i sin @

i ... 8e&raction inde o& the mediume.g. 1.0 &or air* u/ to 1.5 &or oil3

... hal& o& the o"!ecti,e o/ening angleA/erture3

193

HAiry dis%s: o/tical "asic structures



97

194

#eceleration /hase shi&t3 o& light "y /assingthrough an o"!ect

htt/:www.microsco/yu.comtutorials!a,a/hasecontrast/hases/ecimensinde.html

195

Khler Illumination

; was esta"lished to guarantee o/timalillumination o& o"!ects.

his illumination is usually also a/rereuisite &or di&&erent contrast methods/hase contrast* di&&erential inter&erencecontrast3 to wor%* as the necessary

com/onents are o/timied &or Khlerillumination.

In order to get a Khler illumination* youha,e to &ocus the o"!ect &irst* then youclose the &ield iris* so that !ust the middle/art o& the ,iew &ield is illuminated i&necessary you ha,e to center the light/ath3 and then the ,ertical /osition o& thecondensor is ad!usted so that the "orderso& the &ield iris a//ear shar/ and &ocussed.



98

196

Correct and wrong illumination

Correct Koehler illumination

&ield iris at wrong ,ertical /osition o& the condensor

light /ath not centered

197

S/eci&ications o& o"!ecti,es

Magni&ication 10* 20* 60;3: totalmagni&ication is gi,en "y o"!ecti,emagni&ication and occular magni&ication ormagni&ication o& the lens in &ront o& the CC#camera3

Immersion medium: air* oil D or waterEigh magni&ication 60 D 1003 with oil orwater

additional &eatures e.g. suita"ility &or&luorescence due to low auto&luorescence o&the glass: 9eo&luar..3

contrast &eatures: e.g. /hase contrast e.g.h13

correction o& lenses &or chromatica""erations e.g. A/ochromat3

correction o& lenses &or /lanarity o& &ocuso,er the ,iew &ield lan3



99

198

Contrast enhancement in transmitted light

microsco/y

4. #i&&erential7Inter&erence contrast9ormars%i3: Ma%ing use o& light/olariation and its change througho"!ects to generate a contrast

2. hase contrast:Ma%ing use o& the/hase o& light when

it /asses an o"!ect

1. Staining o& structurese.g. nucleus: "lue withhematoylin* antigen:"rown with immuno7histochemistry3

199

hase contrast

+nstained o"!ects such as cellsslow down the light the /hase o&the /assing light3 "y λ. hasecontrast rings in the o"!ecti,e canaccelerate the light* which doesnot /ass through cells "y λ* theresulting di&&erence o& f λ causesan inter&erence* which leads to

contrast enhancement. λ

f λ

htt/:www.microsco/yu.comtutorials!a,a/hasecontrasto/ticaltraininde.html



100

200

hase contrast II

he /hase contrast rings o& the o"!e%ti,e and the condensor ha,e to match each other indiameter and ha,e to "e concentric. In addition the distance "etween them has to "ecorrect which is the case at the Khler illumination3 D this is es/ecially im/ortant &orhigher magni&ication o"!ecti,es. It is stated on the o"!e%ti,e which /hase contrast ring hasto /ut in at the condensor e.g. h1* h2...3.

201

/hase contrast III

wrong /hase contrast ring

/hase contrast rings not centered

correct Koehler illumination and/hase contrast

htt/:www.microsco/yu.comtutorials!a,a/hasecontrastmicrosco/ealignmentinde.html



101

202

Illumination scheme o& an in,erted microsco/e

condensor with /hasecontrast rings

Kno" to ad!ust the,ertical /osition o& the

condensor

&ield iris

screws &or centering thelight /ath

203

#i&&erential7Inter&erence7Contrast #IC39ormas%i Contrast3

Be&ore reaching the condensor* the light is /olaried and/asses a dou"le /rism Lollaston rism3* where it is s/litinto two "eams with di&&erent directions and/er/endicular wa,es. hese "eams /ass the sam/le*where they are altered in intensity and /hase etc. he"eams are &ocussed "y the o"!ecti,e. In the &ocal /lanethere is a second dou"le /rism* which com"ines the

"eams again. A&ter that* the "eams are de/olaried again.here"y the "eams that ha,e "een altered di&&erentially inthe sam/le can inter&ere with each other D and thisinter&erence results in changes o& the intensity and thecolour.

he outcome is a /reudo7threedimensional image.



102

204

Com/arison

hase Contrast D #i&&erential Inter&erence

Ee$a cells D same ,iew &ield

htt/:www.microsco/yu.comtutorials!a,a/hasedicmor/hinde.html

205

luorescence Microsco/y

>am/le: ri/le7luorescence7la"eled

endothelial cells:8ed: Actin7ilamentsla"eled with halloidin

(reen: Mem"ranes #i-7C3

Blue: 9uclei #AI7stainingo& #9A3



103

206

Basics o& &luorescence microsco/y

luorescent sam/les areecited with light o& ana//ro/riate wa,elengththrough the o"!ecti,e3* theemitted &luorescence iscollected again "y theo"!ecti,e and is guided to adichroic mirror* whichse/arates the ecitation light&rom the emitted&luorescenceF the latter

/asses an emission &ilter andis detected "y eye or "ya//ro/riate detectors such ascameras3

Interacti,e )eiss7utorials: htt/:eiss7cam/us.magnet.&su.edututorials

207

Scheme o& a &luorescence microsco/e



104

208

$ight sources &or &luorescence ecitation

1. Con,entional light sources:

7 mercury lam/s:

7 Yenon7lam/s:

$># $ight Sourceslight7emitting diodes: semiconductor de,ices3

&rom: htt/:eiss7cam/us.magnet.&su.edu



105

Metal Ealide $am/s

&rom: htt/:eiss7cam/us.magnet.&su.edu

211

$aser light sources

2. $aser $ight Am/li&ication "yStimulated >mission o& 8adiation3:

(i,e !ust discrete wa,elengthslines3 D thus the choice o&ecitation light is limited andde/ends on the laser ty/e.

Ar7laser: main lines at 6?? nm and

516 nm* and 65?3Ee9eon: 564 nm* 44 nm

+7$aser: 605 nm

iolet laser diodes: 605 D 620 nm

Ad,antages o& laser light:

7high uality /arallel light "eams37good &or scanning

7high intensity



106

212

luorescence ilter Cu"es

he &ilter cu"e consists o&:

1. >citation &ilter: !ust thecorrect ecitation lightwa,elength3 /asses the &ilter

2. #ichroic mirror: is re&lecti,e&or the ecitation light "uttransmittent &or the emissionlight the emitted &luorescence3

D se/arates ecitation &rom&luorescence light

4. >mission &ilter: &ilters theemitted light so that !ust thecorrect wa,elength e.g. indou"le &luorescence3 reachesthe detector

sample

213

Characteristics o& &luorescence &ilter sets

>citation &ilterBand ass3

>mission &ilterBand ass3

emission &ilter

ecitation &ilter

dichroicmirror



107

>am/le o& a "and/ass &ilter G nomenclature

214

215

#ual"and &ilter sets: Simultaneous o"ser,ationo& two di&&erent &luoro/hores e.g. >(#s8ed3

>citationdichroic mirror

>mission



108

216

Monochromators as light source

A con,entional light source e.g. a Yenon lam/3 is s/lit "y amonochromator e.g. a di&&raction grid3 to the s/ectral colours Dto /roduce light o& a &reely de&ina"le wa,elength 420 D <00 nm3.his can "e used instead o& a &ied ecitation &ilter.

-ne ad,antage is that this technology allows switching "etweendi&&erent ecitation wa,elengths within &ew milliseconds. his can"e im/ortant &or ecitation ratio imaging e.g. ura72 Calciumimaging etc.3

+ 420 nm3

8ed <00 nm3electronicallyad!usta"le grid

olychrome &romI$$ hotonics

217

#etection o& the emitted &luorescence7 isually ,ia the occular o& the microsco/e

7 "y a CC# camera usually cooled to reduce the electronic noise3.he /hotons o& the &luorescence hit a light sensiti,e chi/ e.g. outo& 1400 1040 /iels3* where electrons are released de/endenton the intensity o& the &luorescence. >ach chi/ can resol,e a gi,enintensity range D e.g. 25 grey ,alues &or a ?7"it camera or 6000grey ,alues &or a 17"it camera.

he images can "e shown on a com/uter monitor and sa,ed on acom/uter

7 "y /hotomulti/lier tu"es MOs3: used o&ten &or scanningde,ices such as con&ocal laser scanning microsco/es. he gain,oltage3 o& the M de&ines the sensiti,ity electrons released &ora gi,en num"er o& /hotons that hit the detector3. -&ten more

Hnoisy than CC# camera images. A,eraging is used to smooththe images good images ta%es a"out 6 sec D while CC# reuire

!ust a"out 100 msec3.

7 old &ashioned: &ilm camera and sensiti,e &ilm e.g. 100 ASA3



109

218

>am/le &or a &luorescence microsco/y

e/eriment7 Cells trans&ected with &luorescent &usion /roteins o& a transcri/tion&actor and its inhi"itor a//ear in the cytosol3F

7 addition o& le/tomycin B $MB3 to "loc% nuclear e/ort. his leads toaccumulation in the nucleus indicating continuous nucleo7cyto/lasmicshuttling

219

>am/le &or a &luorescence microsco/ye/eriment II

0 20 40 60 800

1

2

3

4

5

min

c y t o s o l i c / n u c l e a r f l u o r

.

Fluorescence was quantified in the nucleus and in the cytosol of the same cellafter different time points > shows the kinetics of nuclear accumulation by thechange of the cytosolic/nuclear ratio.

The data were fitted by nonlinear regression analysis (single exp. decay) –

leading to the half time of the nuclear import process.



110

220

rotocol o& an immuno&luorescence staining

iation: 15 min 6P ara&ormaldehyd

4 5 min mit BS wash 50mM ris7ECl /E<.6* 150 mM 9aCl* 0.1Priton3

Bloc%: 1 h at 8 with 4P BSA in BS

Incu"ation with 1. A": anti7Iκ B ra""it/olyclonal* sc74<1 Santa Cru31:400 in BS4P BSA* o,er night at6NC or 1 h at 4<NC3.

2 5 min wash with BS* 1 5 minwith BS

Incu"ation with Alea6?? goat anti7ra""it 1:2000 in BSBSA: 1 h at 4<NC

4 5 min wash with BS* 1 5 minwith BS

Mounting

221

Com"inations o& transmitted light and &luorescence

A3 directacuisition with"oth light sources

B3 Se/arate acuisition o& &luorescence and /hase contrast and mergeor "lending e.g. with ImageJ or other so&tware3

I7$a" So&tware

ImageJ so&tware



111

222

Interacti,e Microsco/y #emonstrations

9ery recommenda+le:

%ttp:micro.magnet.fsu.edu

1. Optical resolution:htt/:www.microsco/yu.comtutorials!a,aimage&ormationairynainde.htmlhtt/:www.microsco/yu.comtutorials!a,alightandcolorre&ractioninde.html

2. <D%ler 'llumination:htt/:www.microsco/yu.comtutorials!a,a%ohlerinde.html

5. %ase s%ift of lig%t +y an o+ecthtt/:www.microsco/yu.comtutorials!a,a/hasecontrast/hases/ecimensinde.html

6. %ase contrasthtt/:www.microsco/yu.comtutorials!a,a/hasecontrasto/ticaltraininde.html

htt/:www.microsco/yu.comtutorials!a,a/hasecontrastmicrosco/ealignmentinde.html

. O+ecti)es ,it% adusta+le ,orking distancehtt/:www.microsco/yu.comtutorials!a,aa"errationscorrectioncollarinde.html

223

Con&ocal $aser Scanning Microsco/y C$SM3

hotomulti/lier

con&ocal /inhole

dichroic mirror

-"!ecti,e

7Motor

Scanner

$aser

ro"lem in con,entional microsco/y: light* which comes &rom outside o& the &ocal /lanea"o,e or "elow3 gets to the detector or eye3 and is registered as "lur* which decreases theuality o& the image

Solution: A /inhole iris3 is /laced into the light /ath at a /osition* where it can "loc% out7o&7&ocus light. By that means an o/tical section is imaged with ,aria"le thic%ness starting witha//ro. 0.? Qm3 de/ending on the diameter o& the /inhole.

+sually high uality ecitation light is needed &or that e.g. coherent laser light with /arallellight "eams3. he result is a ,ery shar/ image without any "lur &rom out7o&7&ocus light with a

slightly higher resolution than with con,entional e/i&luorescence microsco/y.



112

Con&ocal microsco/y remo,es the "lur&rom thic%er o"!ects

htt/:eiss7cam/us.magnet.&su.edututorialso/ticalsectioningcon&ocalwide&ieldinde.html

-/tical sectioning and 4#7/ro!ections

225

Acuisition o& a H7stac% image slices along the 7ais3 allows reconstruction o& a 4#7/ro!ection* whichcan "e shown as animation

7stac%

4# rendering/ro!ection



113

226

S/ectral imaging

H>mission &inger /rinting: emission scan o& amicrosco/y sam/le Hlam"da stac% o& images3 at agi,en ecitation wa,elength e.g. with )eiss $SMM>A systems or with $eica con&ocal microsco/es;3

Alternati,e: >citation scan at a constant emissionwa,elengthF e.g. using a monochromator lightsource3

Com"inations o& ecitation and emission &inger/rinting e.g using &ilter wheels3

Increases the num"er o& mar%ers to "e measured in

/arallel Can "e used to discriminate &luoro/hores with

o,erla//ing s/ectra

Can "e used to discriminate s/eci&ic &luorescence&rom auto&luorescence

8esol,ing s/ectral in&ormation on a /iel7"y7/iel "asis

$eica conce/t

)eiss M>A conce/t

227

lambda7stac%

Spectral curve of aregion of interest

$eica Con&ocal Microsco/e CS S2: Monochromator in &ront o& the detector A-BS: Acousto7-/tical Beam S/litter instead o& dicroic mirror3

M>A System o& )eiss: 42 M7detectors e,ery 10.< nm600 D <20 nm3: simultaneous wa,elength analysis.

S/ectral Imaging Con&ocal Microsco/ywith >mission Cur,e Analysis3



114

Zimmermann et al.

(FEBS Letters 2003) Sample with overlappingfluorophores

Emission curves separated

into 8 channels (left) or

2 channels (right)

Equation matrix for the

channel signals based on

reference intensities in the

channels (GFPn and YFPn)

and the unknowncontributions of the

fluorophores

Unmixed fluorescence

(pseudo-coloured)

1 2 4

6 5

< ?

1

2

S/ectral imaging eam/le I: C* ( and =

229

htt/:eiss7cam/us.magnet.&su.eduarticless/ectralimagingintroduction.html



115

S/ectral imaging eam/le II: strongly

o,erla//ing dyes

230

S=-Y (reen nucleus3* Alea luor6?? con!ugated to /halloidin&ilamentous actin networ%3* and-regon (reen 516 con!ugated to goatanti7mouse /rimary anti"odiestargeting mitochondria3.

htt/:www.in,itrogen.comsiteusenhomeroducts7and7Ser,icesA//licationsCell7

Analysis$a"eling7Chemistryluorescence7S/ectraiewer.html

In,itrogen S/ectra iewer

Se/aration o& s/eci&ic &luorescence &rom auto7&luorescence "y s/ectral imaging

231



116

>am/le &or >mission inger/rinting on a )eiss

$SM510 M>A: Se/aration o& ( and =

Acquisition of a

reference lambda

stack for the first

fluorophore (GFP)

500 510 520 530 540 550 560Emission wavelength (nm)

0

50

100

150

200

250

Intensity

Obtain the spectral emission curve for the first fluorophore and

repeat the procedure for the second fluorophore

GFP

500 510 520 530 540 550 560Emission wavelength (nm)

0

50

100

150

200

250

Intensity

YFP



117

+nmiing o& a mied sam/le

(7Actin and =7mem"ranes3

Emission stack

Unmixed image

235

image with mied signals &ordi&&erent mar%ers

+nmiing o& signals in /athology sam/les

auto&luorescence

Bright&ield dis/lay

Shown with the 9uance7So&tware &rom Cam"ridge 8esearch ` Instrumentation3



118

236

H8ealtime con&ocal microsco/y* S/inning dis%

con&ocal microsco/y with 9i/%ow7dis%s3

gentle scanning less "leaching good &or sensiti,e li&e cells

#etection o& the signal with a CC#7camera

htt/:eiss7cam/us.magnet.&su.edututorialss/inningdis%yo%ogawainde.html

237

Com/anies &or con&ocal microsco/es

)eiss: htt/:www.eiss.de

$eica: htt/:www.leica.comwww.confocal-microscopy.com

• Nikon: http://www.instrumente.nikon.de/

• Olympus: http://www.olympus.de/microscopy/



119

238

Multi/hoton $aser Scanning7Microsco/y

A uantum /hysical /henomenon is used: at ,ery high lightdensities using /ulsed lasers* a"out @00 nm in&rared light3/ac%ages o& 2 or more /hotons occur !ust in the &ocal /lane U3.hese ha,e the same energy as single /hotons o& higher energyshoerter wa,elength* e.g. 650 nm3. hus these /hoton /ac%agescan ecite a &luoro/hore* which emits then at &or instance 520 nmmitted wa,elength is horter than the ecitation light wa,elength U3.

An im/ortant ad,antage is that the @00 nm light has a mucherdee/er /enetration into tissue a//ro. 1 mm3* while con,entionalecitation can image !ust down to 0.25 mm. Another ad,antage is a

reduced o,erall "leaching e&&ect* as ecitation /hoton /ac%agesoccur !ust in the &ocal /lane.

239

Multi/hoton $aser Scanning7Mi%ros%o/ie II

con,entional ecitation17hoton cone o&ecitation light3

27hoton ecitation:only a s/ot o&

ecitation



120

240

S/ecial luorescence Microsco/y echniues

1. 0!A: luorescence 8eco,ery A&ter hoto"leaching

2. 0$': luorescence $oss in hoto"leaching

5. 0!ET: luorescence 8esonance >nergy rans&er

6. 0$'4: luorescence $i&etime Imaging Microsco/y

. 0'S8: luorescence In Situ Ey"ridiation

241

8A: luorescence 8eco,ery A&ter hoto"leaching

An image is ta%en D then a region o& the cell is "leached "y high laser intensity* &ollowed "ya time series o& images a&ter "leaching. Brie&ly a&ter "leaching the region is signi&icantlydar%er and then the &luorescence intensity increases again &luorescence reoo,ery3 due todi&&usion o& molecules into the "leached area. he %inetics o& reco,ery de/ends on thedi&&usion coe&&icienceF the etent o& reco,ery the /lateau to which the &luorescencereco,ers3 is a measure o& the o,erall mo"ility the &raction o& mo"ile molecules ,ersusmolecules immo"ilied* e.g. to the cytos%eleton3

8A in the cytosol:

8A at the mem"rane

Non linear regression analysis

y = span (1-e-kx) + bottom



121

in,erse 8A with no,el &luorescent/roteins

243

rotocol: 8A analysis on )eiss $SM510 Ca/ture an image o& the whole cell "e&ore "leaching #e&ine a "leaching scan region and may"e in addition another scan region that is not

"leached3 er&orm a time series with 1 scan /re"leach* a"out <0 iterations o& "leaching with 100P

laser /ower and then 507100 scans o& the "leach region and also the non7"leached controlregion i& you s/eci&ied one37 a good time resolution can only "e o"tained i& !ust the small"leach region and may"e the control region3 is scanned 7 and not the whole cellFa,eraging o& 2 or 6 scans reduces the electronic noise and leads to "etter uanti&ications.

Ca/ture an image o& the whole cell a&ter the 8A time series with the same conditions asthe /re"leach image D &or calculating the total loss o& &luorescence.

I& you want to sa,e dis% s/ace: etract the 8A region and sa,e !ust this region instead o&

the whole image It is recommended to use the LCI ,ersion o& ImageJ &or analysis: =ou can o/en the $SM7

&iles with the "uilt7in &eature which also allows o/ening the time ,alues o& the imageseries3. Measure the mean &luorescence in a control region or &or the whole cell &or "oth the/re"leach and the /ost"leach images and calculate the loss o& o,erall &luorescence due tothe "leaching in the region o& interest this is necessary &or o"taining correct reco,ery,alues &or the "leach region3.

Im/ort the 8A7image seuence* de&ine a measurement region and a//ly the Hintensity,ersus time /lot /lug7in D this will draw a gra/h o& the 8A cur,eF clic%ing the list "utton*shows a list o& the numerical ,alues the &irst 6 /arameters are dimension and /osition o&the region* the rest are the &luorescence intensity ,alues3.

Co/y the &luorescence raw data &rom the list to the corres/onding column o& an >celtem/late



122

244

bottomespan y kx

+−×= −

)1(

Calculate the di&&erence o& mean &luorescence &rom the "ac%ground and normaliethe &luorescence ,alues to 100P &or the initial &luorescence.

#i,ide the /ercent ,alues "y the correction &actor calculated &rom the total loss o&&luorescence e.g. i& total &luorescence decreased &rom 1 to 0.@ then di,ide themean &luorescence o& the 8A regions &or each time ,alue "y 0.@ to com/ensate&or the loss in total &luorescence3. A similar com/ensation can "e o"tained "ynormaliing the 8A &luorescence ,alues to the control scan region that was not"leached. his method also com/ensates more eactly &or the "leaching e&&ect inthe course o& scanning o& the time series this scanning7de/endent "leachinge&&ect is o//osed to the reco,ery o& &luorescence in the "leach region due todi&&usion o& non7"leached molecules in the "leach region3. his dynamiccorrectionX gi,es a somewhat "etter estimation o& the cur,e and the %inetics o&the reco,ery3 D "ut leads in /rinci/le to results that are ,ery similar to the cur,eo"tained with the constant correction &actorX "y calculating the total loss in&luorescence "ased on the intensities o& the images that were ca/tured "e&ore anda&ter the 8A7time series3

or non7linear regression analysis cur,e &it o& the data to a single e/onentialassociation algorithm3: Co/y the data to a &itting /rogram such as (ra/hadrism3 and /er&orm the &itting with a "ottom to s/anX algorithm:

$I: luorescence $oss in hoto"leaching

; to determine the dynamic shuttling o& molecules "etween di&&erentcom/artments o& the cell

A certain com/artment A e.g. the cytosol3is re/etiti,ely "leached "y the laser D andthe &luorescence decrease in a di&&erentcom/artment B is monitored "y time la/semicrosco/y. Molecules that shuttle &rom Bto A are "leached in A thus thecom/artment B gets dimmer when there isa dynamic distri"ution o& molecules"etween A and B. 0

20

60

0

?0

100

120

0 2 6

cytosol

nucleus



123

$I to determine a nuclear e/ort signal and anucleolar localiation signal

nuclear FLIP(bleach in nucleus outside nucleoli)

0 100 200 300 400 500 600 7000

25

50

75

100

125nuclear

nucleolar

sec

9B inducing %inase

truncated 9IK without the e/ort seuence:

CS: luorescence Correlation S/ectrosco/y

; to determine di&&usion coe&&icients and interactions "etween molecules.he sam/le is illuminated "y the laser at a ,ery small s/ot* the mo,ementso& molecules in this s/ot in and out3 cause &luorescence &luctuations* whichare analyed "y correlation &unctions



124

248

8>: luorescence 8esonance >nergy rans&er

Microsco/y>nergy can "e trans&erred "etween two &luoro/hores when theyare ,ery close to each other closer than 10 nm3 and when theemission cur,e o& one the energy donor3 o,erla/s with theecitation cur,e o& the other one the acce/tor3. his trans&er o&energy does not ha//en ,ia /hotons U3 "ut "y a di/ole7interactiona uantum /hysical /henomenon disco,ered "y heodor 0Drsterin 1@63. As a result the donor &luoro/hore &luorescence "ecomeswea%er and the acce/tor &luorescence increases.

he 8> e&&ect decreases with the th /ower o& the distanceF thedistance o& hal& maimal energy trans&er is called drster 7#istance8

0

&or C and = it is a//roimately 5 nm3. As the e&&ect isusually not detecta"le anymore at a distance higher than 10 nm itis ideally suited &or monitoring macromolecular interactions/rotein7/rotein or /rotein7#9A3. By that means* not only theinteraction "y itsel& can "e detected* "ut also the location o& theinteraction and its dynamics.

249

rinci/le o& luorescence 8esonance >nergy rans&er

excitation

emission

nm

Donor Acceptor

> \ 8 0 8 0

G r3 und

8 0 \ κ 2 × Jλ3 × n76 × WT1 × @<0

80 ; rster7#istancer ;.. real distanceκ ;.. -rientation &actorJλ3 ; s/ectral o,erla/n ; re&racti,e indeW ; Wuantum yield o& &luor.

DonorAcceptor

ecitation

no FRET FRET

donor fluor.

(CFP)

acceptor fluor.

(YFP)



125

250

A//ro/riate luoro/hore airs &or 8>

luoro/hore air Comments

C0 F0 (ood com"ination &or normal 8> microsco/y using Eg7lam/s as light source and s/ecial &ilters. C is /oorlyecitated "y Ar7lasers* "ut: good ecitation "y "lue laserdiodes

G0 =0 B has in&erior &luorescence /ro/erties

=0 "s!ed-)ariants -riginal #s8ed is !ust &luorescent as tetramer* showscom/le maturation characteristics with green &luorescentintermediates and tends to aggregateF #s8ed2 is a dimer.

he new monomeric #s8ed wor%s ,ery &ine with (.

=0 F0 Are ,ery di&&icult to se/arate with &ilters "ut can "e used in$IM and with s/ectral analysis3

=0 Cy5 or Alexa 6 Anti"odies can "e directly la"eled with Cy4 or eui,alent Alea dye and gi,e 8> with a ( chimera to which they"ind.

0'TC T!'TC Classical 8> /air &or la"eled /roteins e.g. anti"odies3

Alexa 633 Alexa 6

&Cy5 Cy(

alternati,es as la"eling dyes su/erior to IC and 8IC3

251Tet-la+el

=0(797κ B

>citation o&( 6?? nm3

8>

(7>mission 512 nm3

et7>mission 560 nm3

#9A

8> can "e used to monitor /rotein7#9Ainteractions

0.0

0.2

0.4

0.6

0.8

1.0

1.2

480 500 520 540 560 580 600

nm

r e l a t i v

e f l u o r e s c e n c e

GFP-NFkB (Em)

Tet (Em)

Tet (Ex)

Spectral overlap

S/e%tral analysis o& a miture: Increase inacce/tor &luorescence indicates 8>

s/ectra o& donor and acce/tor (797κ Band et7la"eled #9A* res/ecti,ely3



126

252

X Y

Donor Acceptor

FRET

1 nm

ECFP and EYFP-Scans

0,0

0,2

0,4

0,6

0,8

1,0

1,2

350 400 450 500 550 600

nm

r e l a t i v e f

l u o r e s c e n c e

EYFP (Em)

EYFP (Ex)

ECFP (Em)

ECFP (Ex)

Spectral o)erlap

decreasein #onor>mission

increase in Acce/tor>mission

rster #istan 8 0 &or >C and >=: ca. 5 nm 50P8> >&&iien3: no 8>7Signal "eyond 10 nm.

8> can "e a//lied to ,isualie the interaction

o& signaling molecules in li,ing cells

253

Overview of FRET-Microscopy Techniques

1. Acuisition with 8> &ilter set donor ecitation and acce/toremission3: ro"lem: coecitation o& the acce/tor at the donorwa,elength &alse /ositi,es

2. Acuisition o& a ratio image o& acce/tor &luorescence at donorecitation and donor &luorescence at donor ecitation27ilter 8> Microsco/y: Just wor%s when there is eual e/ression

o& donor and acce/tor e.g. in &usion /rotein* "iosensors3

4. 47ilter 8> Microsco/y

6. #etermine the %inetics o& donor &luorescence "leaching this isslower in the /resence o& a 8> acce/tor* as /art o& the "leachingenergy is trans&ered to the acce/tor3

5. #onor reco,ery a&ter acce/tor /hoto"leaching

. &luorescence li&etime microsco/yD $IMF &luorescence li&etime o& thedonor decreases in the /resence o& a 8> acce/tor



127

254

ECFP and EYFP-Scans

0.0

0.2

0.4

0.6

0.8

1.0

1.2

340 360 380 400 420 440 460 480 500 520 540 560 580 600nm

r e l a t i v e

f l u o r e s c e n c e

S/ectral crosstal% o& donor and acce/tor

ro+lems:

1. Co-excitation of t%e acceptor at t%e "onor-excitation ,a)elengt% H#on-0!ET-0luorescence in t%e ra,-0!ET c%annel

2. Signal-o)erlap of donor into t%e acceptor c%annel H #on-0!ETfluorescence in t%e ra,-0!ET c%annel

>citation window

o& donor

>mission window

o& acce/tor

raw 8>7channel:

#onor >citation G Acce/tor >mission

255

8atio o& donor emission and acce/tor emission at the ecitationwa,elength o& the donor

ECFP and EYFP-Scans

0.0

0.2

0.4

0.6

0.8

1.0

1.2

350 400 450 500 550 600nm

r e l a t i v e

f l u o r e s c e n c

e

EYFP (Em)

EYFP (Ex)

ECFP (Em)

ECFP (Ex)

excitation

emission-1 emission-2

$imitations:concentration de/endentdonor and acce/tor ha,e to

colocalie com/letely

!ust use&ul &or 8>7"iosensors with co,alentlin%age "etween donor andacce/toreual7molar e/ression and100P colocaliation3

image \ >mission2 : >mission1

2 ilter78> Microsco/y 8atio Imaging3



128

256

4 Images are ta%en under constant camera settings3:1. #onor e.g. C7ecitation and emission3*

2. Acce/tor e.g.=7ecitation and emission D this signal is not a&&ected "y 8>

4. 8>7ilter raw 8>: C7ecitation and =7emission3.

A normalied 8> signal image3 can "e calculated "y using correction &actorso"tained with single stained sam/les:

FRETc = IFRET - corrCFP x ICFP – corrYFP x IYFP

corrC0 : ca. 0.59

corr F0 : ca. 0.18

C = neg. control C7= /os. control

47ilter 8> Microsco/y

8> microsco/y eam/le

257

sample "onorc%annel

Acceptorc%annel

0!ETc%annel

corr.factor

corrected0!ET

C alone 100 0 0 0. 0

= alone 0 100 20 0.2 0

non7"oundC G =

100 100 ?0 0

"ound C7= 100 100 10 ?0

corrected FRET = IFRET - corrCFP x ICFP – corrYFP x IYFP

#onor Acce/tor corrected 8> normalied 8>

9ormalied 8>normalied to di&&.e/ression le,els3:

=

×

neg.control

sam/le



129

258

; this is slowed down in /resence o& a 8> acce/tor

time series of images

EYFPECFP

436 nm

FRETECFP

436 nm

time series of images

476 nm 476 nm

C7rotein alone

C7 and =7rotein

8> Microsco/y "y analying the %inetics o&

donor "leaching

259

offset.A0 += −kt e y

single exponential decay

y... Fluor. Signal

A0... starting signal

k... decay constant

t... time

offset... final value

Fluorescencehalf time Tau:τ

= 0.69/k

FRETeff . E = 1 - (τ without / τ with Akzeptor.)

ro"e mit 8>

ro"e ohne 8>

advantages:

concentration independent

donor and acceptor don‘t have to

colocalize completely

Limitation: requires external control,

difficult to obtain a FRET-image

#onor7"leaching%inetics



130

260

#onor reco,ery a&ter acce/tor "leaching: An image o& the donor in the /resence o&the acce/tor is ta%en* then the acce/toris "leached /artially3* &ollowed "yacuisition o& a second donor image

AcceptorDonor

AcceptorDonorFRET

8> Microsco/y "y acce/tor "leaching and

monitoring donor reco,erydo not use &or C =3

261

isualisation o& "iochemical reactions "y 8>microsco/y e.g. /hos/horylations3

#etection o& the auto7/hos/horylation o& >(7rece/tor on yrosine residues using(7>(8 &usion /rotein and Cy47la"eled anti77yr anti"odies

donor reco,ery a&ter acce/tor /hoto"leaching techniue3

GFP

erb2 -P

Cy3

( 8atio image



131

262

$IM: luorescence $i&etime Imaging Microsco/y

he li&etime o& donor &luorescence usually in the nanosec. range3 isreduced in /resence o& a 8> acce/tor.

his li&etime can "e determined "y a s/ecial ,ariant o& microsco/y. +sually a/ulsed or a modulated laser is used &or ecitation. he &luorescence decayime #omain3 or the /hase shi&t reuency #omain3 o& the emissioncom/ared to the ecitation is a measure o& the &luorescence li&etime.

$IM image&luor. image

263

8>7Biosensors I

Cas/ase 47Biosensor:

A/o/tosis Acti,ation o& cas/ase 43 is detected with a C7= &usion/rotein in which C and = are se/arated "y a cas/ase 4 clea,age site.

Lithout a/o/tosis: 8>* with a/o/tosis: no 8>

7;#>#;7 8>

no 8>

Cas/ase 4C =



132

264

-ther eam/les &or 8>7Biosensors

KA Acti,ity sensor: C and = se/arated "y a KA7su"strate seuenceand a 167474 domain* which "inds /hos/ho7serine o& the KA su"strate

domain.

C =

8>

=CKA

Calcium7Biosensor: Ca2G7sensiti,e Calmodulin and a Ca2G Calmodulin7"indingM14 domain are s/liced "etween C and = additional localiationseuences can "e added D e.g. signal /e/tide and >8 retention seuence3. Achange in the calcium concentration leads to a change in the con&ormation o&the lin%er and thus to an alteration o& the 8> signal.

PKA-substrate 14-3-3

C =Ca2G

Calmodulin M14Ca2G

C

=8>

265

#igital Image Analyses

-&ten used: 'mageI scienti&ic reeware: htt/:rs"we".nih.go,i! 3

#i&&erent o/erations can "e /er&ormed: contrast enhancement*smoothing* "ac%ground su"traction* measurement o& &luorescence

intensities; D and &urther more e,en math with images can "e done e.g.di,iding one image "y another oneF each /iel ,alue is di,ided "ythe corres/onding /iel ,alue o& the second /icture D at the same/iel coordinates3



133

266

#i,ision o& imageswith ImageJ

i8> lugin &or ImageJ



134

8> analysis with sel&7written ImageJ macro

neg. control

sam/le

0

2

6

?

10

12

16

9egati,eControl

IKK1GMyc IKK2G Mycsam/le 1 sam/le 2

269

In Situ Ey"ridisation ISE3

htt/:www.cytochemistry.netIn]situ.htm

htt/:osiris.rutgers.edusmmin]situ]hy"ridiation.htm

; &or s/eci&ic detection o& #9A or m89A seuences. A la"eled #9A7 or 89A is hy"ridied to the target seuence in situin the cell or the tissue3 and detected

A//lications:

1. #etection or semi73 uanti&ication o& m89A

2. #etection o& #9A7seuences in chromosomes e.g. translocations*mutations* loss o& genes;3



135

270

rinci/le o& ISE

- a labeled“ probe (e.g. DNA, labeled with Biotin-dUTP by Nick-Translation or anoligonucleotide, labeled by terminal deoxynucleotidyl transferase, TdT) diffusesinto the cell and hybridizes with the target sequence. Addition of formamide to thehybridization buffer lowers the specific hybridization temperature, so that at 37°Conly specific target sequences are bound. The probe is then detected viafluorescence (fluorescence in situ hybr., FISH) or by enzyme activity (e.g. HRPand colour reaction).

IC

271

ISE: Ad,antages and #isad,antages o& ,arious /ro"es

Probe type Advantages Disadvantages

DNA (double strand) Easy to use

Subcloning unnecessary

Choice of labeling methods

High specific activity

Possibility of signal amplification

(networking)

Reannealing during hybridization

(decreased probe availability)

Probe denaturation required,

increasing probe length and

decreasing tissue penetration

Hybrids less stable than RNA probes

DNA (single strand) No probe denaturation needed

No reannealing during hybridization

(single strand)

Technically complex

Subcloning required

Hybrids less stable than RNA probes

RNA Stable hybrids (RNA-RNA)

High specific activity

No probe denaturation needed

No reannealing

Unhybridized probe enzymatically

destroyed, sparing hybrid

Subcloning needed

Less tissue penetration

Oligonucleotide No cloning or molecular biology

expertise required

Stable

Good tissue penetration (small size)

Constructed according to recipe

from amino acid data

No self-hybridization

Limited labeling methods

Lower specific activity, so less

sensitive

Dependent on published sequences

Less stable hybrids

Access to DNA synthesizer needed

more recently: BAC /ro"es can "e o"tained &rom collections3: high sensiti,ity &or single co/y genes;

&rom: eldman* 8S* Meyer* JS* and Wuener* $ 1@@<3. rinci/les o& 9euro/sycho/harmacology . Sunderland* MA: Sinauer Associates* Inc. ages 41745.



136

272

>am/les &or ISE

luorescence In Situ7Ey"ridiation onMeta/hase7chromosomes

#etection o& a target7m89A in cryo7or /ara&&in sections

"etection ,it% 55

"etection ,it% alkal. %osp%atase

ISE with inter/hase nuclei

273

m89A7ISE rotocol

Cells are fixed ,it% fres%ly made 6J formalde%yde in GS* p8 .6 for 1 min atroom temperature. All solutions s%ould +e made in 4olecular Giology gradeultrapure ,ater &no !#ase(. Kear glo)es at all times and use sterile disposa+lepipets and tips.

After rinsing in GS &5 L 1/ min. eac%(* cells are permea+ilied ,it% /.J Triton L-1// in 1L GS for 1/ min at 6oC.

Cells are t%en rinsed in GS &5 L 1/ min. eac%( and t%en in 2L SSC &1 L min.(. 1// ng of nick translated pro+e &containing digoxigenin d;T( and 2/ ug of

competitor E. coli t!#A per co)erslip are dried do,n in a Speed 9ac &Sa)ant(.T%is is a good starting place +ut you may %a)e to titrate your specific pro+e.

1/ Ml of deionied formamide is added to t%e dried "#A.

T%e pro+e and t!#A are denatured +y %eating for 1/ min at N/oC. T%e pro+e isc%illed on ice immediately. 1/ Ml of 8y+ridiation +uffer &2/J dextran sulfate 6L SSC( is added to t%e

denatured pro+e so t%at t%e final concentrations in t%e %y+ridiation mixtureare ngml of pro+e* 1 ugml of E. coli t!#A* 2L SSC* and 1/J dextran sulfate.

2/ Ml of %y+ridiation mixturepro+e is placed onto eac% co)erslip. Co)erslips are in)erted onto a slide and sealed ,it% ru++er cement and

incu+ated in a %umid c%am+er for 1 %rs. at 5oC. After rinsing in 2L SSC/J formamide at 5oC* 2L SSC and 1L SSC at room

temperature for 5/ min. eac%* t%e co)erslip containing cells are incu+ated in 6LSSC/.2J GSA2ugml anti-digoxigenin anti+ody for / min. in a %umidc%am+er at room temperature in t%e dark.

Co)erslips are t%en rinsed in 6L SSC &1 L 1 min.( at room temperature* 6LSSC/.1J Triton L-1// &1 L 1 min.(* and 6L SSC &5 L 1/ min. eac%(.

Co)erslips are mounted in fluorescence mounting medium.

4odified from: Iimne-=arcPa* $. and ".$. Spector. 1NN5. Cell 5* 6-N.



137

Su/erresolution Microsco/y I

S>#: Stimulated >mission #e/letion

274

A second laser de/letion laser3 Htrims the ecitation s/ot /oint7s/read &unction* S3 to asmaller sie. 8esolution a//r. ?0 nm.

htt/:eiss7cam/us.magnet.&su.eduarticlessu/erresolutionintroduction.html

Su/erresolution Microsco/y IIStructured Illumination Microsco/y SIM3

275

A second laser de/letion laser3 Htrims the ecitation s/ot /oint7s/read &unction* S3 to asmaller sie. 8esolution a//r. ?0 7 100 nm.

A %nown /attern is /ro!ected into theimage /lane at di&&erent angles andinter&eres with sam/le structures*creating Moirj /attern. Su/erresolutionin&ormation can now "e ca/tured "y themicrosco/e &rom these structures "ymathematical algorithms.&rom www.eiss.de3

normal image SIM image



138

Su/erresolution Microsco/y

7 "y single molecule detection

276

STO!4: Stochastic O/tical ! econstruction 4icrosco/y using single &luorescent molecules

A$4: hotoacti,ated $ocaliation 4icrosco/y

8esolution: a//r. 40 nm* "ased on statistical calculation o& the center o& a (aussian it o& asingle molecule. 8euires a sensiti,e camera e.g. >MCC#: >lectron7multi/lying charge7cou/led de,ice cameras3 D and some so&tware* "ut no s/eci&ic hardware

htt/:eiss7cam/us.magnet.&su.eduarticlessu/erresolutionintroduction.html

277






microsco/y

&low analysis ACS3




139

278

0lo, CytometrySome contents are ins/ired "y luorescence S/ectrosco/y in

Biological 8esearchX

"y 8o"ert . Mur/hy

0lo, Cytometry D Measuring /ro/erties o& cells in &low

0lo, Sorting D Sorting se/arating3 cells "ased on /ro/erties measured in

&low

D Also called 0luorescence-Acti)ated Cell Sorting &0ACS(

#e&initions:

279

Basics o& low CytometryBasics o& low Cytometry

•Cells in suspension

•flow in single-file

through

•an illuminated volume

where they

•scatter light and emit

fluorescence

•that is collected,

filtered and

•converted to digital

values

•that are stored on a

computer

Fluidics

Optics

Electronics7 low Cytometry* low Analysis3.

7 low Sorting* luorescence Acti,ated Cell Sorting* ACS



140

280

htt/:/ro"es.in,itrogen.comresourceseducation

281

9eed to ha,e cells in sus/ension &low in single&ile through an illuminated ,olume

In most instruments* accom/lished "y

in!ecting sam/le into a s%eat% fluid as it/asses through a small 507400 Qm3 orifice

luidics



141

Lhether &low will "e laminar can "e determined &romthe !eynolds num+er

282

luidics II

Lhen conditions are right* sam/le &luid &lows in a central corethat does not mi with the sheath &luid

his is termed $aminar flo,

is the mean &luid ,elocity in SI units: ms3

# is the diameter m3

_ is the dynamic ,iscosity o& the &luid aks or9ksm3

is the %inematic ,iscosity \ _ 3 ms3

is the density o& the &luid %gm3

W is the ,olumetric &low rate ms3

A is the /i/e cross7sectional area m3

Lhen 8e 2400* &low is alwayslaminar

Lhen 8e 2400* &low can "etur"ulent

283

Flow Cell

InjectorTip

Fluorescencesignals

Focused laserbeam

Sheathfluid

Purdue University Cytometry Laboratories

8ydrodynamic0ocusing



142

284

The figure shows the

mapping between the

flow lines outside and

inside of a narrow

tube as fluid

undergoes laminar

flow (from left to

right). The fluid

passing through crosssection A outside the

tube is focused to

cross section a inside.

V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

Eydrodynamic ocusing

285

V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

Notice how the ink is focused

into a tight stream as it is drawn

into the tube under laminar

flow conditions.

Eydrodynamic ocusing II

Sam/le container and the sheath &luid container are /ut under de&ined air/ressure. $aminar &low is maintained and hydrodynamic &ocussing isachie,ed3 "y a /recise control o& the /ressure di&&erence "etween the sam/lecontainer* the sheath &luid container and the atmos/here

>am/le: ocusing o& in% "ylaminar &low into a ca/illary

In &low cytometry:



143

286

H.B. Steen - MLM Chapt. 2

Flow

through

cuvette

(sense inquartz)

low Cham"er

#i&&erent ty/es are /ossi"le*

a commonly used ty/e:

287

PMT

PMT

PMT

PMT

DichroicFilters

Bandpass Filters

Laser

1

2

3

4

Flow cell

original from Purdue University Cytometry Laboratories;

modified by R.F. Murphy and J. Schmid

SSC

FSC

-/tics: (eneral scheme



144

288

orward Scatter SC3Lhen a laser light source isused* the amount o& lightscattered in the &orward

direction along the same aisthat the laser light is tra,eling3

is detected in the for,ardscatter c%annel

he intensity o& &orwardscatter is mainly /ro/ortional

to the sie and sur&ace

/ro/erties o& cells or other/articles3

289

;sually not a sensiti)e 4T detector&+ecause t%e lig%t intensity is %ig%(

Q +ut rat%er a p%otodiode detector*,%ic% can +e set in log1/-increments &E-1* E/* E1...(* and alinearity gain factor &1./ Q N.NN(.

A +locking +ar pre)ents t%e directlaser lig%t from %itting t%e detector.

A predefined 0SC-Kert is often usedas t%res%old to discriminate+et,een cells and dust particles

orward Scatter#etector



145

orward scatter threshold to ignore de"ris andsmall /articles or cells

290

291

Lhen a laser light source isused* the amount o& lightscattered to the side/er/endicular to the ais thatthe laser light is tra,eling3 isdetected in the side or N/o

scatter c%annel

he intensity o& side scatter ismainly de/endent onsu"cellular structures e.g.granules* ,esicles;3

Side ScatterSSC3



146

292

90 Degree Light Scatter (SSC)

FSC Detector

SSC detector

Laser


N/° scattered lig%t&side scatter( isdetected ,it% ap%otomultiplierdetector* ,%ere t%esensiti)ity can +e setin 9olt. Amplificationscale can +e linear orlogarit%mic.

-/tics: luorescence Channels

dichroic mirrors

"and/ass &ilters



147

294

Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm

457350 514 610 632 488 CommonLaserLines


luoro/hores

295

Sorting o& cells

488 nm Laser

+-chargedelectrodes

Sortedcells

Fluorescence

FSCSensor

+ - control unit

-

Purdue University Cytometry Laboratories; modified

GatingFSC

FL1



148

296

297

Sorting Mode

>clusion7Mode: dro/lets containing non target cells are dismissede,en i& they contain target cells high /urity* may"e lower yield

8eco,ery7Mode: dro/lets containing target cells are collected* e,en i&they also contain non7target cells high yield* may"e low /urity.

Single Cell Mode: only dro/lets with single target cells are collected

high /urity* may"e low yield* high counting accuracy.

I& there are 2 or more cells in one dro/let*and the dro/let contains target and nontarget cells* then there is a Hsorting con&lict*which has to "e sol,ed "y de&ining ana//ro/riate sorting mode:

arget7Cell

9on7target cell



149

298

-,erla/ o& &luorescence signals and HCom/ensation

299

HCom/ensationLhen cells are la"eled with 2 &luoro/hores e.g. IC and>3* there might "e a signal crosstal% "etween the detectionchannels de/endent on detector ,oltage settings3. his can"e com/ensated in the &ollwing way: measuring a sam/lecontaining only &luoro/hore 1: i& there is crosstal%* you seean ele,ated intensity in "oth channels D using thecom/ensation control o& the so&tware* you can "ring the&luorescence signal in the wrong channel down to the"ac%ground &luorescence. Lith a sam/le stained only with&luoro/hore 2 you do the same &or the other channel. hiscom/ensation is usually !ust ,alid &or the detector settingsat which it was set.

com/ensation &or &luoro/hore 1com/ensation &or &luoro/hore 2



150

300

Com/ensation II

"e&orecom/ensation

a&tercom/ensation

true dou"le /ositi,es can"e determined

301

ACS7(ra/hs

Eistograms: the intensity o& onechannel is di,ided into classeso&ten 1026* 7ais3 and the&reuency o& He,ents cells3scored into these 1026 classes.

#ot lots: 2 di&&erent /arameters

are /lotted on the 7 and y7aisFeach cell is a s/ot on this gra/haccording to its /arameterintensities.

counts

0

$1

$2

Correlation "etweenhistograms anddot/lots



151

302

ACS7(ra/hs II

4#7lots: he &reuency o& e,entsmeasured &or 2 /arameters is illustrated"y a third ais 7ais3 in a 4# manner.

Contour lots: he &reuency o& e,entscells3 in ,arious areas o& a 2#7/lot is,isualied "y lines re/resenting eual/ro"a"ility similar to contour lines o&mountains on a ma/3

$1

$2

$1

$2

#ensity lots: he &reuency o& e,entscells3 in ,arious areas o& a dot /lot iscolour coded to highlight the /ea%s

$2

303

Statistics and H(ating

8egions: can "e de&ined indi&&erent &orms /olygons*elli/ses..3. hese can "e used &orstatistics o& cells &alling into acertain region D "ut also &or Hgating that means re!ectingcertain cells inside or outside3 acertain region &or analysis or

data acuisition3 D or &or instance&or sorting o& cells.

Mar%er: +//er and lower limits inhistograms &or uanti&ication

Wuadrants: s/lit the 2# gra/h in 6regions* "y the coordinates o& the/oint where 2 ais intersect. Allows &ast and sim/le statistics.

8egion 81: @.4P

olygon78egion3

Mar%er M1: <.5P

+$ u//er le&t3: 0.0P+8 u//er right3: 2@. ??P$$ lower le&t3: 0.6P$8 lower right3: @.66P



152

304





analyses o& molecular interactions


microsco/y

&low analysis ACS3


305

Analytical A//lications o& low Analysis

$eu%ocyte analyses

henoty/ing o& cells C#7Mar%er3

Immuno&luorescence stainings

cell cycle analyses #9A7content3

Chromosome analyses

roli&eration assays Brd7+ incor/oration3

A/o/otosis assays Annein * d* JC13

Calcium &lu7measurements

etc.



153

306

$eu%ocyte analyseshenoty/ing o& cells

C#?7/ositi,e 7cells

C#67/ositi,e 7cells

307

cell cycle analyses "y staining o& #9A with I

10000 200 400 600 800

G0G1

s G2M

DNA Gehalt2N 4N

G2M

G1

s

G0

a/o/toticcells

(0 (1

(2 M

S

ropidium-'odide 0luorescence of permea+ilied cells after digestion of !#A:fluor. depends on "#A content

di/loidChromosome content

dou"led di/loid



154

308

rotocol o& a ro/idium7Iodide Staining

Adherent cells:trypsinized, suspended in medium + 10% FCS, centrifuged (1000 rpm, 5 min), Pellet

suspended in PBS (1 ml)

Suspension cells:

Centrifuged (1000 rpm, 5 min), Pellet suspended in PBS (1 ml)

Fixation with EtOH:Pipet cell suspension into 2.5 ml absolute EtOH (final concentration approx. 70%) - or vortex

the suspension at half speed while adding the EtOH) – to prevent clustering of cells during thefixation. Incubate on ice for 15 min (or over night at –20°C).

Alternative fixation with paraformaldehyde:

Pipet the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r.t.

Staining:

Pellet the cells at 1500 rpm for 5 min, Suspend the pellet in 500 µl PI-solution in PBS:

50 µg/ml PI from 50x stock solution (2.5 mg/ml), 0.1 mg/ml RNase A, 0.05% Tritin X-100Incubate for 40 min at 37°C

Add 3 ml of PBS, pellet the cells (1500 rpm, 5 min) and take off the supernatant

Suspend the pellet in 500 µl PBS for flow analysis

(you can also leave about 500 µl of the diluted staining solution on the pellet and suspend the

cells in this solution > less loss of cells when you take off the sup.) – the rest of the stainingsolution does not interfere with the flow analysis.

Flow analysis:

Approximate settings (on FACSort):FL1: 570 V log. (e.g. if you want to detect GFP)

FL2: 470 V linear

309

Chromosome Analyses



155

310

Analysis o& roli&eration

Brd+7la"eling o& S7hase Cells3

IC7anti7Brd+ luor.

ro/idium!odid luor. I3

Cells are cultured &or a gi,entime in medium containingBromodeoy7+ridin Brd+3. hisnucleotide analogon incororatesinto newly synthesied #9A o&cells in S7/hase3 D and can "edetected with anti7Brd+anti"odies e.g. IC la"eled3.

311

Staining o& a/o/totic cells with JC71

JC-1 (5, 5´, 6, 6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazol-carbocyanine iodide)

is a dye, which incorporates in to mitochondrial membranes, where the fluorescence

depends on the membrane potential !n normal cells (with intact mitochondrial

membrane potential) it builds a""re"ates and emits mainly red fluorescence, in

apoptotic cells (where the membrane potential brea#s down) it occurs in monomers

and emits mainly "reen fluorescence $his can be detected by flow analysis or

microscopy



156

312

rotocol o& a JC71 Staining o& A/o/totic Cells

JC71 is /re/ared as a 1000 stoc% solution in #MS- 5 mgml3.

or the staining o& adherent cells it is diluted in medium to 5 Qgml with,orteing during the dilution to /re,ent the &ormation o& /reci/itates3F the JC71 containing medium is added to the cells* &ollowed "y incu"ation &or 10 minat 4<°C or 8 &or 15 min3.

Su"seuently the cells are washed twice with BS* try/sinied* sus/ended in500 Ql BS and analyed "y &low analysis.

Sus/ension cells lym/hocytes3: sus/end 1:1 with 10 Qgml JC71 in medium&inal conc.: 5 Qgml3

A//roimate detection settings on ACSort:

$1: 40 log3

$2: 410 log3

Com/ensation : $17<P $2 und $27<6P $1

313

#etection o& a/o/totic cells "y I7staining o&/ermea"ilied cells cell cycle analysis3

ragmented #9A emitslower &luorescence thencells with the normaldi/loid #9A content D this

HSu"7(0(1 /o/ulationre&lects a/o/totic cellswith &ragmented #9A 3ata late stage o& a/o/tosis3



157

314

#etection o& a/o/totic cells with Annein

luorescence7la"eled Annein : "inds to/hos/hatidylserine* whichis normally on the innerlea&let o& the mem"rane*"ut which is &li//ed to theoutside during a/o/tosis

#iscrimination "etween necrotic andapoptotic cells

I7/osAnnein7neg.: necroticI7negAnnein7/os.: early a/o/toticI7/osAnnein7/os.: late a/o/totic* ornecrotic with large holes in the mem"ranewhere Annein can get through3

315

Calcium lu #etermination "y low Analysis

Cells are stained with a calcium sensiti,e &luoro/hore I9#-71 or"etter luo63F a&ter a stimulus* the %inetics o& the &luorescence signalis measured monitoring calcium in&lu3.



158

316

Sources

urdue ;ni). Cytometry $a+oratorieshtt/:www.cyto./urdue.edu

8esearch Institute o& Scri//s Clinic: htt/:&acs.scri//s.edu

In,itrogen utorialshtt/:www.in,itrogen.comsiteusenhomesu//ortutorials.html3

B# utorials: htt/:www."d"iosciences.comsu//orttrainingit&]launch.!s/

I. aul !o+inson urdue +ni,.3

!o+ert 0. 4urp%y Carnegie Mellon +ni,.* itts"urgh3

0lo, Cytometry and Sorting* 2nd ed. M.8. Melamed* .$indmo* M.$. Mendelsohn* eds.3* Liley7$iss* 9ew =or%* 1@@0 D

a"ge%'rt: 4$4 0lo, Cytometry: 'nstrumentation and "ata Analysis

M.A. an #illa* .9. #ean* -.#. $aerum* M.8. Melamed* eds.3* Academic ress* $ondon* 1@?5 D a"ge%'rt: 9"$4

317

Com/anies o&&ering &low analysis eui/ment

Becton7#ic%insonhtt/:www."d.com

Bec%man7Coulterhtt/:www."ec%mancoulter.com

Milli/ore:htt/:www.milli/ore.com

Accuri now /art o& B#3htt/:www.accuricytometers.com

artec: htt/:www./artec.com



159

318

ree So&tware: lowing So&tware 2htt/:www.&lowingso&tware.com

lowing So&tware 2

319

8egions in histograms

Wuadrants



160

320

Cell cycle analysis with lowing So&tware

321

• #e&ine 4 histogram regions E1* E2* E4:(0(1* S and (2M7/hase* res/ecti,ely3

• Acti,ate the region control tool: Cell Cycle

• #e&ine the (2 /ea% multi/lier and /ea% widthright clic% in the cell cycle window3

• Choose acti,e control a&ter right clic%ing3

• Create statistics "y right clic%ing into thehistogram window3

• Create a Stat.$ist "y right7clic%ing into theStatistics window

• Ctrl79 loads the net &ile* ad!usts the E17E4regions automatically and calculates the cellcycle /hases E17E43



161

issue Cytometry: Wuantitati,e Image Analysis o&

microsco/y sam/les

htt/:www.tissuegnostics.com

Single7cell recognition e.g. "ased on #AI7nuclear &luorescence3 generation o& cell mas%s &oruanti&ication o& signals e.g. in di&&erent &luorescence channels3 scattergrams can "e deri,ed similar to&low cytometry &or the cells in their original tissue en,ironmentU3 (ating and statistics are /ossi"le

Similar e,aluations can "e done withImageJ using automatic threshold and the HAnalye /articles &eature.

issue Arrays &or uantitati,e com/arison o& sam/les

CA PINNormal

Pat. #1

Pat. #2Pat. #3

Pat. #4Control

Gingiva

Cystectomy

Example of a

prostate tissuearray

> equal staining conditions for all samples



Multi7/arallel coordinate /lots &or ,isualiation

o& se,eral /arameters in /arallelCan "e done with reeware Mondrian:

htt/:www.theusrus.deMondrianinde.html3

-ne e,ent e.g. one cell3 is re/resented "y a line lin%ingse,eral y7ais &or the di&&erent /arameters e.g.&luorescence signals3F a H/o/ulation can "e selectedand is highlighted also &or the other /arameters. hedata density can "e reduced using a so called al/ha7&actor3 to o"tain "etter ,isi"ility o& numerous data/oints.

he density o& lines e,ents3 can "e ,isualied in colour7coded manner eam/le: 107/arameter /arallelcoordinate /lot generated with Mat$a"3 &ast intuiti,e,isualiation o& multi/arameter data sets

1 2 4 6 5 < ? @ 10

methods cell biology 2015

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