This document provides an overview of flow cytometry and fluorescence-activated cell sorting (FACS). It describes flow cytometry as a technique for measuring physical and chemical characteristics of cells as they flow in a fluid stream, allowing for single cell analysis. FACS extends this by using fluorescence to identify cell characteristics and sort cells into separate collections based on these characteristics. The key components of a flow cytometer are described as lasers, optics including filters and detectors, fluidics to hydrodynamically focus cells, and electronics to convert optical signals to digital data. Applications including cell phenotyping, apoptosis analysis, and cell cycle analysis are discussed. Cell sorting and quantitative analysis of cell cycle phases are also summarized.

1. Fluroscence- Activated
Cell Sorter (FACS)
FROM: Nidhi Parikh
M.Sc. Biochemistry

2. What is Flow Cytometry?
• Cytometry refers to the measurement of
physical/chemical characteristics of cells or other biological
particles.
• Flow Cytometry is the process whereby such
measurements are made upon cells/particles as they pass
through a measuring apparatus (hopefully in single file)
suspended in a fluid stream.

3. WHY???
• Flow Sorting (Flow Cytometric Cell Sorting) extends flow
cytometry with the additional capacity to divert and collect
cells exhibiting an identifiable set of characteristics either
mechanically or by electrical means (Flow Cytometric
Analysis).
• FACS - Fluorescence Activated Cell Sorting? FACS is a
trademark of Becton Dickinson Immunocytometry Systems
(BDIS). All FACS instruments are BDIS systems, but not
all cytometers are FACS.

5. What Is Flow Cytometry?
• Flow ~ cells in motion
• Cyto ~ cell
• Metry ~ measure
• Measuring properties of cells while in a
fluid stream

6. The Many Parts of Flow
• Experimental design
• Sample preparation
• Choosing the proper instrument
• Setting up the instrument
• Collecting the proper data
• Interpreting the data
• Graphics presentation and publication
• Sorting

7. Instrumentation

8. Flow cell
Injector
Tip
Fluorescence
signals
Focused laser
beam
Sheath
fluid

9. How The Flow Cell Works
• The cells from the sample tube are
injected into the sheath stream
• Flow in a flow cell is laminar.
• Hydrodynamic focusing pushes the cells
to line up single file along their long
axis.
• The shape of the flow cell provides the
means for hydrodynamic focusing.

11. Sample Differential
10 psi
10.2 psi
10 psi
10.4 psi
10 psi
10.8 psi
Difference in pressure between sample and sheath
This will control sample volume flow rate
The greater the differential, the wider the sample
core.
If differential is too large, cells will no longer line up
single file
Results in increase in multiple cells passing
through the laser at once. No more single cell
analysis!

12. Incoming Laser
Sample
Sheath Sheath
Sheath
Sample
Sample
Core
Stream
Low Differential High Differential
Laser Focal Point

14. 488 nm laser
+-
Fluorescence Activated Cell Sorting
Charged Plates
Single cells sorted
into test tubes
FALS Sen
Fluorescence detec

15. Cytometry vs. Flow Cytometry
Cytometry
• Localization of
antigen is possible
• Poor enumeration
of cell subtypes
• Limiting number of
simultaneous
measurements
Flow Cytometry.
• Cannot tell you
where antigen is.
• Can analyze
many cells in a
short time frame.
• Can look at
numerous
parameters at
once.

16. Laser
Light
488nm
FL2 PE
SSC
FSC
Fluidics
Electronics
Computer
FL3 PerCP, Cy5
FL1 FITC
A cytometer consists of:
Light detectors

17. What Happens in a Flow Cytometer?
• Cells in suspension flow single file
past
• a focused laser where they scatter
light and emit fluorescence that is
filtered and collected
• then converted to digitized values
that are stored in a file
• Which can then be read by
specialized software.
Interrogation
Fluidics
Electronics
Interpretation

18. Laser interrogation and signal processing
followed by sort decision: sort right, sort
left, or no sort.
Electronic delay until cell reaches break
off point. Then the stream is charged : +
or -.
Charged droplets deflected by electrostatic
field
from plates held at high voltage (+/-3000
volts).
Besides tubes can sort onto slides or
multi-well plates.
The nozzle/flow cell is vibrated by a
transducer (converts electrical energy into
mechanical energy) so it produces a
stream breaking into droplets.
Flow Cytometry Sorting Schematic

19. 1. LASER
2. OPTICS
3. FLUIDICS
4. ELECTRONICS
FACS COMPONENTS

20. LASER
Light amplification by stimulated emission of radiation
• Lasers can provide a single wavelength of light
(monochromatic)
• They can provide milliwatts to watts of power
• Also provide coherent light
• All help to create a stable and reliable signal
Coherent: all emmiting photons have same wavelength, phase and
direction as stimulation photons

21. LASER

22. Emission of Radiation

23. Absorption & Emission of Radiation

24. FLUORESCEIN TEXAS RED
PHYCOERYTHRIN ALLOPHYCOCYANIN
PROPIDIUM IODIDE RHODAMINE
WAVELENGTH (NM)
EXCITATION OR EMISSION SPECTRA OF VARIOUS
FLUROPHORES

25. 1. BEAM SPLITTERS
2. DICHROIC MIRRORS
3. LONG / SHORT PASS
FILTERS
4. BAND PASS FILTERS
OPTICS

27. FORWARD SCATTER
1. LIGHT SCATTER USED IS LOW
ANGLE
2. SENSITIVE TO CELL SIZE AND
SURFACE AREA
3. LIVE / DEAD DISCRIMINATION

28. FORWARD SCATTER
FSC
Detector
Laser Beam

29. SIDE SCATTER
1. LIGHT SCATTER USED IS 90o
2. SENSITIVE TO INTERNAL
STRUCTURES
BEST ANALYSIS USING FORWARD
AND SIDE SCATTER TOGETHER;
ANALYSIS OF HETEROGENEOUS
POPULATIONS

30. Side Scatter
FSC
Detector
Collection
Lens
SSC
Detector
Laser Beam

32. • Some Information Can Be Obtained
• FSC Correlates With Cell Size
• SSC Correlates With Internal Complexity
• To Distinguish Between 2 Cell types
– A. Size Has To Be Different OR
– B. Internal Complexity i.e amount of granules
• If These Two Parameters Are The Same, Then
No Distinction Can Be Made
Limitations with light scattering

33. FLUIDICS
1. SAMPLE FLUID
2. SHEATH FLUID
3. FLOW CELL
4. NOZZLES, 70, 100, 400 um

34. ELECTRONICS
1. PHOTOMULTIPLIER TUBES
(PMTs)
2. PHOTODIODE
3. MACINTOSH G3 COMPUTER

35. Detectors
• There are two main types of photo
detectors used in flow cytometry
 Photodiodes
o Used for strong signals, when saturation is a
potential problem (eg. FSC detector)
 Photomultiplier tubes (PMT)
o More sensitive than a Photodiode, a PMT is
used for detecting small amounts of
fluorescence emitted from fluorochromes.

36. Electronics
• Detectors basically collect photons of
light and convert them to current
• The electronics must process that light
signal and convert the current to a
digitized value that the computer can
graph
• Thus convert optical signal into
electronic signal

37. Creation of Voltage pulse

38. Quantification of Voltage pulse

40. Cell sorting using FACS

41. ADVANTAGES OF FACS
1. PRECISION
2. SENSITIVITY
3. NO PHOTOBLEACHING
4. VOLUME
5. SINGLE CELL ANALYSIS
6. SAMPLING STATISTICS
7. SORT CELLS IN SEPARATE TUBES

42. • Cell size.
• Cytoplasmic granularity.
• Cell surface antigens (phenotyping).
• Apoptosis.
• Intracellular cytokine production.
• Intracellular signalling.
• Gene reporter (GFP).
• Cell cycle, DNA content, composition, synthesis.
• Bound and free calcium.
• Cell proliferation (BRDU and CFSE)
• Cell sorting, single cell cloning (clonecyt)
Applications

43. Cell-Cycle Analysis
using FACS

44. CELL CYCLE ANALYSIS
1. FACS IS METHOD OF CHOICE FOR
FAST, ACCURATE DETERMINATION
OF CELL CYCLE DISTRIBUTIONS
2. DETERMINATION OF ANUEPLOIDY
3. % OF S PHASE

46. TUMOR DNA STAINING
SINGLE CELL SUSPENSION
PERMEABILIZE WITH METHANOL
DIGEST DS RNA WITH RNASE
STAIN DNA WITH PROPIDIUM
IODIDE

47. Propidium Iodide

48. G0/G1 = 60%
S = 13%
G2/M = 27%
Cell Cycle Analysis
DNA CONTENT
E
V
E
N
T
S

49. CELL CYCLE ANALYSIS
S
G2/M
G0/G1
T
U
M
O
R
D
I
P
L
O
I
D
G0/G1 = 79.5%
S = 12.7%
G2/M = 7.8%
DNA INDEX = 1.11

50. 1. Measure the forward scatter (FS) and side scatter
(SS) to identify single cells.
2. Pulse processing is used to exclude cell doublets from
the analysis. This can be achieved either by using pulse
area vs. pulse width or pulse area vs. pulse height
depending on the type of cytometer.
3. PI has a maximum emission of 605 nm so can be
measured with a suitable bandpass filter.
Analysis of results

51. DATA PARAMETERS
1. FORWARD LIGHT SCATTER
2. 90o OR SIDE SCATTER
3. FLUORESCENCE EMISSION
Expected results
While running the cytometer, the following plots
should be displayed:
1. Forward and side scatter to identify the cells
2. Pulse shape analysis to identify clumps and
doublets (this can be pulse area vs. pulse width or
pulse area vs. pulse height depending on cytometer)
3. Forward scatter vs. PI signal; PI histogram.

52. Results

53. Results

54. For analysis, first gate on the single cell population using
pulse width vs. pulse area. Then apply this gate to the
scatter plot and gate out obvious debris. Combine the
gates and apply to the PI histogram plot.
Results

55. There are two ways to quantitate the percentage of cells
in each cell cycle phase:
1. By using markers set within the analysis program.
2. By using an algorithm which will attempt to fit Gaussian
curves to each phase. This is available with some flow
cytometry software and is more objective than setting
markers by eye.
Quantification of Percentage of cells in each cell cycle

56. Results

57. Troubleshooting and Tips
Cells should be kept as concentrated as possible to allow
the lowest sample pressure differential to be used.
This will ensure that the core sample stream is as narrow
as possible and give optimal CVs. The CV, or coefficient
of variation, is a measure of spread of the data and is
defined as the standard deviation (sd) divided by the
mean (m) expressed as a percentage (sd/m X 100).

58. Troubleshooting and Tips
Single parameter DNA analysis will not yield any kinetic
information, nor will it be able to distinguish between cells
in very early or late S phase from cells in G1 and G2
phase respectively.
Nor can we distinguish between G2 and Mitotic phase
cells.
For this information, a bromodeoxyuridine (BrdU)
technique should be used or you can combine DNA
analysis with a cell cycle phase-specific marker (e.g.
phospho H3 for mitosis).

59. THANK YOU