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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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