basic introduction and principle - cmurdc.cmu.edu.tw flow cytometry... · © 2016 bd. bd, the bd...

© 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company.

Flow CytometryBasic Introduction and Principle

FACSCanto II & FACSAria Sorter

Kate Chen 陳又楷

Product Specialist

BD Bioscience

[email protected]

Sep 24, 2019

1

© 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company.

FACS: Fluorescence-Activated Cell Sorter

• Developed by Prof. Leonard Herzenberg, Stanford University, 1974, BD 是流式細胞分選儀的先驅


FACSCantoII▪ Introduction of Flow Cytometry

▪ Instrument setup

▪ Applications of Flow analysis

FACSAria Sorter▪ Sorter System

▪ Applications

Topic

3


What is Flow Cytometry?

Flow = Fluid

Cyto = Cell

Metry = Measurement

A variety of measurements are made on cells, cellorganelles, and other objects suspended in a liquid andflowing at rates of several thousands per second through aflow chamber.


Particle Size

• Detection range: 0.5~50m


What Can a Flow Cytometer Tell Us About a Cell?

• Its relative size (Forward Scatter—FSC；前向散射光)

• Its relative granularity or internal complexity

(Side Scatter—SSC；側向散射光)

• Its relative fluorescence intensity


Scatter Light

FSC Sensor

Laser

SSC Sensor

90o

Forward


Cell Line

Cell debris

Aggregation

Scatter Light indicating physical properties of cell


Ex. Lysed Whole Blood

Forward Scatter

Sid

e S

catter

Lymphocytes

Monocytes

Neutrophils

(FSC)

(SS

C)


Flow Cytometry Detection Principle

Cytotoxic T Helper T

CD3

CD4

CD8


Fluorescence Light


Fluorescence intensity


BD FACSCanto IITM


Main Component

Fluidics 液流系統

To introduce and focus the cells for interrogation.

Optics 光學系統

To generate and collect the light signals.

Electronics 電子系統

To convert the optical signals to proportional digital signals,process the signals, and communicate with the computer.


Data Processing

PE

FITC

SSC

FSC

APC

PerCP-Cy5.5

Time

Time

Time

Time

Time

Time1. fluidics

2. optics

3. electronics

Computer operation

Flow Cytometry Detection Principle


Fluidics - BD FACSCanto IITM

FACSCleanFACS Shutdown solution


Sheath Flow

sheath tank

Sample Flow

interior reservoir

interrogation point

waste aspirator

sheath filter

sample injectiontube (SIT)

test tube

flow cell

Fluidics - BD FACSCanto IITM


Sample Flow

Hydrodynamic

Focusing

sheath sheath

sample

Excitation Lasers

Flow Cell


Sample Differential

FACSCantoII flow rate

Low = 10 L/min, Medium = 60 L/min, High = 120 L/min

＊細胞週期實驗請選用低流速上機


Optical

• Excitation optics:

− Lasers

− Filters and mirrors that route the laser light to the fluid stream

• Collection optics:

− Fiber optic cables that direct the emitted light to the appropriate emission block

− Filters that direct the signals in the emission block to the appropriate photomultiplier tube (PMT)


Excitation Optics

488nm

633nm

螢光光束如何分光?


Optical Filters


FACSCanto IITM - Collection Optics


564–606 nm515–545 nm

750–810 nm

483–493 nm

Collection Optics—Octagon

> 670 nm

•接收光源順序：能量弱→強

•反射傳遞：能量折損較低

Blue Laser Signal


Collection Optics—Trigon

650–670 nm

750–810 nm

Red Laser Signal


Excitation and Emission

▪ Use the maximum excitation wavelengths to determinelasers that can be used to excite the fluorochrome.

▪ Use the maximum emission wavelengths to determinefilters and PMTs that can be used to measure the signal.

488

530/30

FITC

Excitation Emission


BD FACSCanto Configuration

488nm Blue Laser

Alexa Fluor 488, FITC, BB515 530/30 502LP

PE 585/42 556LP

NA 610LP

PI, PerCP, PerCP-Cy5.5, BB700 670LP 655LP

PE-Cy7 780/60 735LP

640nm Red Laser

APC, Alexa Fluor 647 660/20 N/A

NA 685LP

APC-Cy7, APC-H7 780/60 735LP

2 lasers, 4 – 2 Configuration


▪ BB515 offers a significantly brighter alternative to FITC

▪ BB515 has less spillover into the PE channel compared to FITC

BD Horizon Brilliant Blue dyes-BB515

FITC

Alexa Fluor® 488

BB515


BB700 is detected in the same channel as PerCp-Cy5.5

29

Tandem Dye: BB core + Cy5.5 like acceptor dyeBrightness: Very BrightEx Max: 485 nmEm Max: 693 nmFilter: same as PerCP-Cy5.5 (e.g. 695/40)


▪ PMTs and preamps convert photons to voltage pulses.

▪ Analog-to-digital converters translate analog signals to proportional digital signals.

▪ Compute area and height for each pulse.

▪ Perform compensation and calculate ratios and width.

▪ An embedded computer interfaces with the computer workstation for data transfer.

Electronics


Creation of a Voltage Pulse

Laser

Time (µs)

Volts

Puls

e H

eig

ht

Pulse Width


Quantification of a Voltage Pulse


Data Storage

39,271

89

675 30,621PE

FIT

C

PE

FIT

C

List-Mode Data

39,27139,271Event 1

Event 2

Event 3

FSC SSC FITC PE

0 60 120 89

10 160 65

30 650 160

675

Time

39,271

22,688

30,621

6,189

Histogram

Dot Plot


Data Display

▪ Linear Scaling

▪ Log Scaling


Linear vs. Log

FL1 Voltage Pulses

Time

Volts

8 V

Time

Volts

4 V

Time

Volts

1.2 V

Time

Volts

0.4 V

Time

Volts

0.1 V

0 V 10 V

1 V 10 V0.1 V0.01 V0.001 V

Pulse Height

Pulse Height

Linear mode

Log mode


Linear v. Log Amplification

▪ Linear amplification is usually used for light scatter parametersand DNA analysis.

▪ Log amplification is used for fluorescence signals with a largedynamic range.





FACSAriaIII▪ Sorter System

▪ Features

▪ Applications

Topic

37


Auto-fluorescence

Non-stain sample

LL

UL UR

LR

Auto-fluorescence After voltage adjustment


Emission Optics

Compensation theory


FITC Spillover

Wavelength (nm)

650nm 700nm500nm 600nm

FITC530/30

Rela

tive I

nte

nsit

y

550nm

PE

585/42


FITC Compensation

To Lower Cluster

Increase %

SubtractedPE

585/42

20.10% of the Signal from

FITC Sensed in PE

Wavelength (nm)

650nm 700nm500nm 600nm

FITC530/30

Rela

tive I

nte

nsit

y

550nm


FITC Compensation

42

650nm 700nm

PerCP-Cy5.5670 LP

500nm 600nm

FITC530/30

Re

lati

ve

In

ten

sit

y

Wavelength (nm)

550nm

PE585/42

To lower cluster, increase value.


Compensation Examples

Correct Compensation Undercompensation Overcompensation

Incorrect Compensation





FACSAriaIII▪ Sorter System

▪ Features

▪ Applications

Topic

44


Applications

▪ Phenotype Analysis(Cell Surface Antigens/Markers)

▪ Intracellular Analysis-- Eg. Cytokines, Signal Transduction molecules…etc.

▪ DNA Analysis-- Eg. Viability, Cell cycle, Apoptosis…etc.

▪ Cell Fuction Analysis-- Eg. Free radicals, Ca2+, Reporter genes…etc.

▪ CBA (Cytometric Bead Array)

-- cytokine detection


▪ Ligand

▪ Receptor

▪ Adhesion molecule

▪ …etc

Phenotype Analysis

Confidential—For Internal Use Only

Lysed Whole Blood Components

Granulocytes

Monocytes

Eosinophils

Basophils

Neutrophils

Lymphocytes

TB NK

T Helper

TCytotoxic

Peripheral White Blood CellsCD45+

CD3+

CD4+ CD3+

CD8+

CD3+

CD3-

CD19+

HLA-DR+

CD3-

CD16+

CD56+

Monocytes

© 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 48

Lymphocyte Subset


▪ Cytokine

▪ Enzymes

▪ Structure Proteins

▪ Intracellular Signaling

Intracellular Analysis


Intracellular Analysis

Permeabilizing solution

Fixation solution


Cell Surface and Cytoplasmic Stain protocol


Detection of Cell Surface and IC Cytokine

CD4 FITC CD4 FITC CD4 FITC


Cell Apoptosis 細胞凋亡

PS

－ Annexin V Apoptosis Assay


Annexin V/PI Double Staining

Early Apoptosis



Apoptosis通常與mitochondria的膜電位(Δψ)去極化有關性

▪ JC-1 = J-aggregate-forming Cationic Δψ sensitive dye

J-aggregates:膜電位Δψ 極化(polarized)時為此型式

JC-1 monomers: 膜電位Δψ (depolarized)去極化時存在此型式

J-aggregates 存於健康細胞，且螢光為FL1及FL2 channels (綠色及橘紅色螢光)

JC-1 monomers通常但非絕對存於Δψ 去極化的apoptosis細胞，且只剩下FL1 (綠色螢光)

－Mitochondria potential change-JC-1 (BD Mitoscreen)


Cell Apoptosis 細胞凋亡－ JC-1(BD Mitoscreen) Jurkat T cell data



－ MitoStatus TMRE/Red


Detergent

Nucleic Acid Dye

Ethanol

DNA Analysis


DNA Dye

N+

C2H2)3

NH2 NH2

N+

C2H5

CH3

C2H5

Propidium 7-AAD


G2M

G0

G1

S

0 200 400 600 800 1000

G0 G1

SG2M

DNA content

Count

2N 4N

Cell Cycle Analysis


(W)

FL2-AFL2-W

Cell cycle study


FL2-W

FL2-A

Cell cycle study


▪ Soluble form protein detection

Application of Flow Cytometry


Cytometric Beads Array (CBA)

72


Simultaneous Analysis of Multiple Cytokines

73


Standard Curves

IL-2 PE MFI:567

IL-2: 125pg/ml

74

© 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 68

Cat# Name Content Size

550285BD Pharmingen™PI/RNase Staining Buffer

The reagent is suspended in a phosphate-buffered solution (pH 7.2) with 0.02% (w/v) sodium azide

100ml

556463BD Pharmingen™ PropidiumIodide Staining Solution

Propidium Iodide Staining Solution(For AnnexinV/PI assay use)

2ml

556419BD Pharmingen™Annexin V

Annexin V-FITC 200 test

556547BD Pharmingen™ Annexin V : FITC Apoptosis Detection Kit I

Annexin V-FITC, PropidiumIodide Staining Solution, Annexin V Binding Buffer

100 test

551302BD Pharmingen™ BD™ MitoScreen (JC-1)

JC-1 dye and assay buffer100 test

564696564697

BD Pharmingen™ MitoStatus TMREBD Pharmingen™ MitoStatus Red

BD Pharmingen™ MitoStatusTMREBD Pharmingen™ MitoStatusRed

25mg100ug

BD Pharmingen Product list

http://www.bdbiosciences.com/ptProduct.jsp?prodId=60493&key=PI&param=search&mterms=true&from=dTable

http://www.bdbiosciences.com/ptProduct.jsp?prodId=12850&key=PI&param=search&mterms=true&from=dTable

http://www.bdbiosciences.com/ptProduct.jsp?prodId=12827&key=apoptosis&param=search&mterms=true&from=dTable

http://www.bdbiosciences.com/ptProduct.jsp?prodId=12865&key=apoptosis~CATALOG_TYPE:Kits/Sets/Pairs&param=search&mterms=true&from=dTable

http://www.bdbiosciences.com/ptProduct.jsp?prodId=110167&key=apoptosis~CATALOG_TYPE:Kits/Sets/Pairs&param=search&mterms=true&from=dTable

http://www.bdbiosciences.com/documents/Mitostatus-Reagents-DS.pdf

http://www.bdbiosciences.com/documents/Mitostatus-Reagents-DS.pdf





FACSAria Sorter▪ Sorter System

▪ Applications

Topic

69



細胞分選儀-基本原理

Nozzle


BD FACSAria Filter Guide


How to form a droplet?

• Amplitude– intensity of the drop drive

• Frequency– number of drops formed per second

• Drop 1– the first disconnected drop

• Gap– Number of pixels between the stream

breakoff and the first drop

nozzle


+ –+

–

+

– Drop generationDrop Charging

Drop Charging

+

+


Drop Charging Delay

+

+

Time


Drop Delay

interrogation point

drop delay

breakoff

Waste

BD FACS™ Accudroptechnology

• Accudrop beads • Diode laser• Camera • Optical filter


• Life or death

• Morphology

• Surface antigens

• Gene expression.

• Cell functions.

• DNA content.

• Specific DNA sequence.

Gating Target cell


Sorting Cells By Surface Markers

• Sorting NK Cells

– CD3 FITC to exclude T cells

– CD56+CD16 PE to include all NK Cells.

R1

R3

R2

分選前的分析分選後結果


Sorting NK Cells for Cytotoxicitity Studies


CD4/CD25 Treg sorting

CD4 PerCP

CD

25

PE 1.35 %

8.94 %

Sort on R1 and R3 sort on R1 and R2


Sorting by Gene Expression

Add viability check.

R2


Rare Cell Sorting tip

• Rare cell sorting usually requires pre-enrichment steps:

– Bring the starting purity to > 5 %

– Ficoll

– Immune Panning

– Magnetic Beads (Positive/Negative)


樣品製備注意事項• 高壓會使buffer的pH值下降, 進而影響細胞的存活. 所以建議於

phenol red -free sample buffer中添加25mM HEPES可使環境的pH值維持一定值.

• sample buffer中的protein含量也會影響分選純度. 若是建議使用5% FCS, 則可改用0.5% - 2% BSA取代以獲得較好結果.

• Collection tube內置放適合細胞存活的cold culture media with higher concentration of FCS.

• Collection tube管壁請先coating 1 - 4% BSA overnight.

• 分選前，樣品先加入viable dye (例如 7-AAD)，以避免分選已死亡細胞。分選後，也應取出一些細胞加入7-AAD再上機確認細胞存活狀況，以改善分選過程中是否有任何疏失之處。

• 樣品必須經35 m濾網以避免塞管


樣品製備注意事項• 儘量多次低速離心以去除細胞碎片與小雜物

• 細胞直徑之考量

• 分選時間不應太長, 即分多管收集細胞. 建議測試不同的分選時間長短(10min, 20min, 或30min)以了解是否會影響細胞的活性與隨後的培養結果.

• Side stream應避免打在管壁上.

• 應使用NA/LE(no azide, low endotoxin) reagent標識細胞. 若是無法找到此類試劑, 則細胞分選下來後應離心清洗多次以去除NA及endotoxin. 離心條件:4oC, 1000rpm.

• 單一clone細胞之培養較mixed population不易, 主要是適合細胞生長所需環境與營養因子太複雜. 以傳統培養hybridoma clones來說, 通常是需要加入feeder cells或是conditioned media. 可試著使用conditioned media加入culture media(5%, 10%或20%)中來加強培養環境.


BD FACSAria III

全世界裝機多的流式分選儀, 自初代2003年推出以來在世界各大研究機構熱銷, 近年來更參與了美國國衛院伊波拉病毒疫苗研究


穩定持續產出高品質期刊Nature.com搜尋結果-AriaIII文獻有1642篇

85


卓越性能—分選後高功能性

2015年，Nature Protocol文章報導經

FACSAria III分選後的細胞>99%是具

有肌原性的， >96%的細胞可以進行終

末端分化。

Nature Protocol 2015:10:1612-1624


來源於人胚胎幹細胞的神經元經BD

FACSAria分選體外培養42天接種在大鼠腦內4周可見明顯的神經細胞生長

Sorted: CD184hi, 30 days post FACS Sorted: CD184lo, 7 days post FACS

Ki67 Map2b Tuj1 Hoechst Synapsin Tuj1 Hoechst Nestin Tuj1 Hoechst Synapsin Tuj1 Hoechst

幹細胞分選後以不同條件培養，分化為不同之神經細胞


循環血腫瘤細胞 (CTC) 與抗藥性間的關聯性

• 單細胞分選 HER2- CTC 觀察單一lineage生長, 隨著培養時間增加, HER2- CTC 轉變為HER2+ CTC

• CTC透過 phenotypeconversion 產生抗藥性

• 探討癌症發展與用藥間的關係


Workflow of Mono-clone Ab Selection

單株抗體篩選平台• 分選融合瘤細胞,大幅縮短傳統限數稀釋單株化的時程

• 單細胞分選後,直接分析 VDJ-H 與 VJ-L DNA 序列,人源化後即可生產單株抗體,應用於疾病治療



單株抗體篩選平台

Workflow of Mono-clone Ab Selection

• 使用分選儀篩選融合瘤細胞• 單細胞分選後


Thank you!

92

陳又楷新加坡商必帝公司生物科學部Product Specialist 產品專員Mail: [email protected]: www.bdbiosciences.com/tw

basic introduction and principle - cmurdc.cmu.edu.tw flow cytometry... · © 2016 bd. bd, the bd...

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