facs aruna
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ANALYTICAL TECHNIQUESIN BIOTECHNOLOGY
LABORATORY(BT1403)
R.ARUNA VIGNESHWARI
96507214001
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FACS
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History
Fluorescence-activated cell sortingis a specialized type of
flow cytometry. It provides a method for sorting a
heterogeneous mixture ofbiological cells into two or more
containers, one cell at a time, based upon the specific light
scattering andfluorescent characteristics of each cell.
It is a usefulscienti
fic instrument, as it provides fast,objective and quantitative recording of fluorescent signals
from individual cells as well as physical separation of cells of
particular interest.
DEFINITION
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FACS is a trademark of Becton Dickinson
Immunocytometry Systems (BDIS).
All FACS instruments are BDIS systems,but not all cytometers are FACS.
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INSTRUMENTATION
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Flow Cell
Injector
Tip
FluorescenceFluorescencesignalssignals
Focused laserFocused laserbeambeam
Sheath
fluid
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488 nm laser
+-
rescence ActivatedSorting
Charged Plates
Single cells sorted
into test tubes
FALS Sensor
Fluorescence detector
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Light Scatter Materials scatter light at wavelengths at which they do not absorb.
If we consider the visible spectrum to be 350-850 nm then small
particles (< 1/10 ) scatter rather than absorb light. For small particles (molecular up to sub micron) the Rayleigh scatter
intensity at 0o and 180o are about the same.
For larger particles (i.e. size from 1/4 to tens of wavelengths) largeramounts of scatter occur in the forward not the side scatter direction -
this is calledMie Scatter(after Gustav Mie) - thus forward scatter is
related to size (at 1-15 microns).
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Hydrodynamic SystemsSample in
Sheath
Sheath in
Laser beam
Piezoelectric
crystal oscillator
FluorescenceSensors
Scatter Sensor
CoreSheath
Signaldirection
Flow Chamber
++ --
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MECHANISM
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Fluorescence Activation Proc
(or Immunofluorescence)
FITCFITC
FITC
FITC
FITC
FITC
Antibodies recognize specific moleculesin the surface of some cells
But notothers
When the cells are analyzed by flow cytometry the
cells expressing the marker for which the antibody
is specific will manifest fluorescence. Cells who
lack the marker will not manifest fluorescence.
Antibodies are artificially conjugated to
fluorochromes.
Antibodies
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SMALL BEAD LARGE BEAD
Frequency Histogram
SMALL BEAD LARGE BEAD
Sample in Sheath
Sheath in
Laser beam
Stream
Charge
+2KV -2KV
Waste
SORT RIGHTSORT LEFT
SORT DECISIONS
Piezoelectric
crystal oscillator
Last attacheddroplet
LEFT RIGHT
Sensors
Sensor
Cell sorting
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PROCEDURE
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- Harvest cells (typically from one 10 cm plate)
- Wash 1X with 5-10 ml of 1X PBS
- Suspend cells in 500ul 1X PBS containing +0.1%
Glucose (keep at 4oC)
- Immediately add 5 ml of cold 70% ETOH (keep at
20oC)
- Mix immediately - Keep at 4oC for between 1hr to 1wk to fix cells
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- Two to three hours prior to FACS analysis - spin cells down - wash 1X with 1X PBS (10ml)
- without adding more PBS, spin again for 2 min sothat the residual PBS can be taken off - after taking off remaining PBS, suspend in 300ul
propidium iodide solution (keep at 4oC) - add 20ul of 10mg/mlRNase - mix and incubate at 37oC for 30-45min. - transfer to FACS tubes and have analyzed.
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The process begins by placing the cells into a flask
and forcing the cells to enter a small nozzle one at a
time
The cells travel down the nozzle which is vibrated atan optimal frequency to produce drops at fixed
distance from the nozzle.
As the cells flow down the stream of liquid, they are
scanned by a laser
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The antibody is bound to a protein that is uniquely
expressed in the cells you want to separate.
The laser light excites the dye which emits a color of
light that is detected by the photomultiplier tube, orlight detector.
By collecting the information from the light (scatter
and fluorescence) a computer can determine which
cells are to be separated and collected.
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The final step is sorting the cells which is accomplished byelectrical charge.
The computer determines how the cells will be sorted before thedrop forms at the end of the stream. As the drop forms, anelectrical charge is applied to the stream and the newly formeddrop will form with a charge.
This charged drop is then deflected left or right by charged
electrodes and into waiting sample tubes.Drops that contain no cells are sent into the waste tube. The
end result is three tubes with pure subpopulations of cells.
The number of cells is each tube is known and the level offluorescence is also recorded for each cell.
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Frequency
distribution
Num
berof
even
ts
Intensity of parameter (e.g. fluorescence)
histogram
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Data generated in flow cytometry is displayed using
Multiparamater Acquisition and Display software
platforms.
Histograms corresponding to each of the parameters
of interest can be analyzed using statistical tools to
calculate percentage of cells manifesting specificfluorescence, and fluorescence intensity.
This information can be used to look at fluorescence
expression within subpopulations of cells in a
sample (gating).
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APPLICATIONS
What can we do with FACS/flow
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What can we do with FACS/flow
cytometry?
1. Cell sorting
2. Analysis of cell size & internal complexity(granularity)
3. Surface phenotyping: Ab against cell surfaceAg, MHC tetramer etc
4. Staining of intracellular protein after cellpermeabilization
: ICS (intracellular cytokine staining) withbrefeldin A/monensin
Detection of phosphorylated protein
5. Cell tracking & proliferation (CFSE)
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6. Analysis of intracellular ion concentration
7. Analysis of reduction/oxidation (redox) potential
8. Cell cycle analysis (by using DNA-binding dye)
9. Apoptosis analysis (annexin V, TUNEL etc)
10. Cytokine secretion assay (& subsequent isolation)
11. Multiplex proteins assay (cytometric bead array)
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