What Can a Flow Cytometer Tell Us About a Cell

1
What Can a Flow Cytometer Tell Us About a Cell?

• Its relative size (Forward ScatterFSC)
• Its relative granularity or internal complexity
  (Side ScatterSSC)
• Its relative fluorescence intensity

2
Properties of FSC and SSC
Right Angle Light Detector ? Cell Complexity
Incident Light Source
Forward Light Detector ? Cell Surface Area

• Forward Scatterdiffracted light
• Related to cell surface area
• Detected along axis of incident light in the
  forward direction
• Side Scatterreflected and refracted light
• Related to cell granularity and complexity
• Detected at 90 to the laser beam

3
Lysed Whole Blood
Which of the three populations is the largest?
How did you make that determination?
Side Scatter
Forward Light Scatter
4
Platelets
Red Blood Cells
4
5
What is Fluorescent Light?
O
HO
? 488 nm
? ? 520 nm
C
Emitted Fluorescent Light Energy
IncidentLight Energy
CO2H
FluoresceinMolecule
Antibody

• The fluorochrome absorbs energy from the laser.
• The fluorochrome releases the absorbed energy by
• vibration and heat dissipation.
• emission of photons of a longer wavelength.

6
Fluorescence
Emitted Fluorescence Intensity ??Binding Sites
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Number of Events
Fluorescent Intensity
7
Emission Spectra
8
Two-Color Cell Analysis
10 4
10 3
CD19 PE
10 2
10 1
10 0
10 0
10 1
10 2
10 3
10 4
CD3 FITC
9
A Cytometer Needs a Combined System of

• Fluidics
• To introduce and focus the cells for
  interrogation
• Optics
• To generate and collect the light signals
• Electronics
• To convert the optical signals to proportional
  digital signals, process the signals, and
  communicate with the computer

10
Basic fluidics of a cell sorter
Flow cell
Nozzle
Sheath
Pressure
Waste
Vacuum
11
Sorting - FACSVantage
Charging Electrode
Sample Injection Tube
Sheath Tube
Vent Tube
Deflection Plates
Collection Tubes
12
Sorting - FACSAria
Deflection Plates
Collection Tubes
13
Parts of a flow cytometer

• Fluidics
• Provide a constant stream of sheath
• Transport the sample to the interrogation point
• Arrange and focus the cells to the laser
  intercept
• Optics
• Focus the excitation light
• Collect the emitted light
• Electronics
• Convert the optical signals into electronic
  signals
• Send the signals to the analysis computer
• Computer
• Display data graphically
• Control instrument settings

14
Fluidics
Air Filter
Air Pump
Flow Cell
SheathRegulator
Waste
Sheath
SampleRegulator
Sheath Filter
Sample
15
Hydrodynamic focussing
Low Sample Pressure12µl/min
High Sample Pressure60µl/min
LaminarFlow
LaminarFlow
Sheath
Sheath
Sheath
Sheath
Sample
Sample
1
16
Summary

• Pressure ( Sheath Pressure) drives the sheath
  buffer through the flow cell, and the higher
  pressure in the sample tube( Sample
  Differential) delivers the sample to the flow.
• In the flow cell, the principle of hydrodynamic
  focussing arranges the cells like pearls on a
  string before they arrive at the laser
  interception point for analysis
• Hydrodynamic focussing cannot separate cell
  aggregates! Flow cytrometry is a technique that
  requires single cell suspensions

17
Basic optics

• Somehow the light from the laser(s) must be
  directed to the measurement cuvette to illuminate
  the cells.
• At the same time, the emitted light must be
  collected to analyse the signals from the cells.

18
Laser
Light Amplification by Stimulated Emission of
Radiation

• Light output from a laser is
• Monochromatic
• Unidirectional
• In phase

Laser
19
Beam shaping and focussing
Focal Length
Laser Beam
BeamExpander
FocusingLens
20 µm
64 µm
32 µm 0 32 µm
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Optics

• Excitation optics consist of
• Lasers
• Fiber optic cables and prisms that route the
  laser light to the fluidic stream
• Collection optics consist of
• 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)

21
Excitation Optics
focusing lens
fiber optic cables
prisms
blue laser (488 nm)
red laser (633 nm)
22
Collection OpticsFilters
Longpass
Bandpass
460 500 540
460 500 540
BP500/50
LP 500
23
Collection OpticsOctagon
530/30
502 LP
670 LP
655 LP
780/60
735 LP
556 LP
585/42
488/10
24
Collection OpticsTrigon
780/60
735 LP
660/20
25
Dichroic mirror efficiency
26
Fluorochromes
27
Review

• We have
• created an illumination region with the
  excitation optics.
• passed the cells precisely through the
  illumination region using hydrodynamic focusing.
• directed the emitted light signals to specific
  detectors (PMTs) by the collection optics.

28
Electronics

• Converts analog signals to proportional digital
  signals
• Computes area and height for each pulse
• Performs compensation, calculates ratios, and
  calculates width
• Interfaces with the computer for data transfer

29
Creation of a Voltage Pulse Analog Signal
30
Conversion of Optical Signals to Proportional
Digital Signals
Analog to Digital Conversion
Signal Out
Digital data to memory
PhotonIn
Voltage In
PMTPower Supply
Sample the pulse 10 mHz
Digitize the pulse 16,384 levels
Levels 01000 Volts
31
Quantification of a Voltage Pulse

• Height maximum digitized value
• Area sum of all height
• Width Area/Height
• Proportional to time of flight

measured
calculated
Pulse Area
Pulse Height
Volts
0
Pulse Width
Time
32
Data Storage
List-Mode Data
PEc
FSC
SSC
FITCr
PEr
Time
FITCc
760
840
638
Event 1
314
85
245
Event 2
Event 3
624
156
612
840
FITC-A
FITC-A
85
400
800
1000
0
0
200
400
600
800
200
600
1000
PE-A
PE-A
245
638
33
Review
Data Processing


34
Flow cytometry - endless applications.

• Phenotyping
• Functional measurements
• Cytokines
• Antigen-specific T cells
• Degranulation
• Cell division

35
SEB stimulation
SSC
No stim
SEB
FSC
CD3
SSC
36
SEB stimulation of PBMCs
No stim
SEB
4
0
INF-?
CD3
CD8
37
Method for detection of cell-specific IL-10
production

• Intracellular staining of IL-10 for flow
  cytometry difficult
• Secretion assay increases sensitivity

37
38
PDC induce IL-10-production in SEB-stimulatedCD4
T cells already at 12 h
SEB 12-hr incubation
SEB 24-hr incubation
SEB 48-hr incubation
pDC
CD11c
39
Fluorescent tetramers/pentamers

• Soluble complex of four or five HLA-peptide
  monomers
• Bind T cell receptors with specificity for that
  particular peptide-MHC-combination
• Allow sensitive and easy detection of antigen
  specific T cells by flow cytometry
• Tetramer positive cells isolated, e.g. by
  immunomagnetic selection

ProImmune.com
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MART-1 pent cells on d12
pMART-1-pent
pCMV-pent
0.41
Donor 3
Median 0.23 of CD8 cells Range0.01-4.17 n9
4.17
Donor 5
Pent.
CD8
41
75 of MART pentamer pos T cells produce INF-?
when stimulated with MART pulsed APCs
MART pent
INF-?
CD8
0.5 MART pos T cells
APC, A2, MART peptide
MART pentamer
No APCs
APC, A2, no peptide
APC, A2, CMV peptide
42
Sorting and expanding pent cells
25.2 of CD8
98.4 of CD8
0.25 of CD8
MART-1 pent
CD8
3.6 x 109 cells
0.5 x 106 cells
43
Peptide-specific degranulation and production of
IFN- ?
HLA-A0201-negativeNo peptide
HLA-A0201-negativeMART-1 10 µg/ml
HLA-A0201-positiveNo peptide
HLA-A0201-positiveCMV-pp65 10 µg/ml
0.67
1.32
2.88
3.10
1.71
2.49
3.87
8.39
8.98
2.65
2.68
7.59
HLA-A0201-positiveMART-1 10 µg/ml
HLA-A0201-positiveMART-1 1 µg/ml
HLA-A0201-positiveMART-1 0.1 µg/ml
HLA-A0201-positiveMART-1 0.01 µg/ml
48.37
9.53
5.96
32.68
5.40
15.83
22.79
4.55
12.23
IFN- ?
14.76
16.69
13.61
CD107a/b
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Isolation protocol DC T
45
Protocol for CD4 T cell stimulation by PDC or
CD11cDC
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(No Transcript)
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Mock
hCMV live
CD123 PDC
CFSE
CFSE
FSC
FSC
CD11c
CFSE
CFSE
FSC
FSC