flow cytometry - molmed · principles of flow cytometry in summary cells in suspension flow in...
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Molecular Medicine Postgraduate School Course Biomedical Research Techniques
FLOW CYTOMETRY
November 1th, 2018
Jaco Kraan, PhD
Dept. of Medical Oncology Erasmus MC Cancer Institute
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FLOW CYTOMETRY
Introduction
Principle of the instrument
Fluidics
Optics
Electronics
Analysis of results
Applications on a flowcytometer
Examples and results
Pros and cons
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Flow Cytometers
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What can Flow Cytometry Do?
Enumerate particles in suspension
Determine “biologicals” from “non-biologicals”
Separate “live” from “dead” particles
Evaluate 105 to 106 particles in less than 1 min
Measure particle-scatter as well as innate fluorescence or 2o fluorescence
Sort single particles for subsequent analysis
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Flow Cytometry Publications/year
YEARS
Pape
rs
Data taken from Medline search using the keywords: “Flow Cytometry”
0
5000
10000
15000
20000
25000
30000
1975 1980 1985 1990 1995 2000 2005 2010
0 25 2697
6652
12310 15180
21944
27042
YEAR
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FLUIDICS Getting the cells in the right place (at the rigth time) using hydrodynamic focusing
J.Paul Robinson http://www.cyto.purdue.edu
The sample is injected into the center of a sheath flow. The combined flow is reduced in diameter, forcing the cell into the center of the stream One cell at a time gets exposed to the laser beam.
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PMT 1
PMT 2
PMT 5
PMT 4
Dichroic Filters
Bandpass Filters
Laser
Flow cell
PMT 3
Scatter
Sensor
Sample
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Optical Filters
Dichroic Filter/Mirror at 45 deg
Reflected light
Transmitted Light Light Source
J.Paul Robinson http://www.cyto.purdue.edu
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Long and short Pass Filters
Transmitted Light Light Source 520 nm Long Pass Filter
>520 nm Light
Transmitted Light Light Source
575 nm Short Pass Filter
<575 nm Light J.Paul Robinson
http://www.cyto.purdue.edu
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From Fluorescence to Computer Display
Individual cell fluorescence quanta is picked up by the various detectors (PMT’s).
PMT’s convert light into electrical pulses.
These electrical signals are amplified and
Each event is designated a channel number (based on the fluorescence intensity as originally detected by the PMT’s) on a 1 Parameter Histogram or 2 Parameter Histogram.
All events are individually correlated for all the parameters collected
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Principles of Flow Cytometry in Summary
cells in suspension
Flow in single-file through
An illuminated volume where they
Scatter light and emit fluorescence
That is collected, filtered and
Converted to digital values
that are strored on a computer
Fluidics
Optics
Electronics
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1 2 3 4 6 7 150 160 170 .. 190
Channel Number
Positive
Negative
Brighter Dimmer Count
1
4
6
Fluorescence picked up from the FITC PMT
Data analysis - 1-parameter histogram
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FITC FL
PE FL
Negative Population
Single Positive FITC Population
Single Positive PE Population
Double Positive Population
Data analysis - 2-parameter histogram or dotplot
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Light Scatter properties (1)
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Light Scatter properties (2)
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Light Scatter properties (3)
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Light Scatter properties (4)
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Side
Sca
tter
0 200 400 600 800 1000
0 20
0 40
0 60
0 80
0 10
00
Lymphocytes
Monocytes
Neutrophils
Platelets
Forward Scatter
Scatter properties (3)
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Fluorescence
λ = 488 nm
Emitted Fluorescent Light Energy
Antibody
Incident Light Energy
Fluorescein Molecule λ ≅ 530 nm
HO
CO2H
O C
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FITC spectral characteristics
FITC PMT BAND PASS
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A TWO COLOR OPTICAL BENCH
FITC PMT
PE PMT
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Spectral overlap in PE channel
FITC PMT BAND PASS
PE PMT BAND PASS
SPILLOVER
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PE spectral characteristics
FITC PMT BAND PASS
PE PMT BAND PASS
SPILLOVER
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INTRA-LASER SPILLOVER
the fluorochrome emission is mainly skewed towards the right
FITC EMISSION
FITC PMT
PE PMT
PE-TR PMT
PE-CY5.5 PMT
PE-CY7 PMT
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Setting electronic compensation for spectral overlap ('color compensation') Using single labeled control cells or beads
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Setting electronic compensation for spectral overlap ('color compensation')
Validate using multiple labelled control cells
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Setting electronic compensation for spectral overlap ('color compensation')
UNCOMPENSATED ! multiple labeled lymphocytes
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Sample Preparation
MUST have a single‐cell suspension with 106 cells/sample ideally!
Always bring a negative control to set voltages and gates.
Bring single‐color controls for compensation, for each fluorochrome used.
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Typical flow cytometry protocol
Cell Surface staining 100µL - 106 cells + 10µL mAb(s)
Incubate for 15’ at RT in the dark
Wash with 2 mL assaybuffer
Centrifuge 10’ 500 g
Fix cells in 1ml PBS/1%PFA
Acquire on FCM
Surface and Intracellular staining Perform cell surface staining
Fix cells in (1%PFA)
Wash with 2 mL assaybuffer
Centrifuge 10’ 500 g
Permeabilize Cells (Triton/saponin)
Wash with 2 mL assaybuffer
Centrifuge 10’ 500 g
10µL mAb(s) and incubate 15’
Wash with 2 mL assaybuffer
Centrifuge 10’ 500 g
Resuspend peelt in 0,5 mL assay buffer and acquire on FCM
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Fluorochrome and mAb selection considerations
Titration of antibodies – to reduce non-specific mAb binding
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Antibody titration
Typical manufacturer’s recommendations: X µL per 1E6 cells (in 0.5 ml).
Background increases with increasing number of Ab molecules.
6 ng/ml 60 ng/ml 300 ng/ml 600 ng/ml unstained
Side Light Scatter
PE-C
D3
inte
nsity
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Fluorochrome and mAb selection considerations
Titration of antibodies – to reduce non-specific mAb binding
Choose bright fluorochromes
Minimize spillover between channels
“Bright” antibodies go on “dim” fluorochromes
Avoid spillover from bright cell populations into channels requiring high sensitivity
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Multicolour Analysis: today up to 15+ colors
- Advantages: Save time, reagents and samples
Exponential increase in information
Identify new/rare populations (<0.05%)
- Problems: Select fluorochrome combinations
Get access to the right instrument
More problems with overlap of emission (Compensation)
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Applications of Flow Cytometry Cel (subset) enumeration (e.g. Lymphocyte subsets, Stem cells)
Celtyping using membrane / cytoplasmatic staining combinations (e.g. leukemia / lymphoma typing)
Cell cycle analysis using DNA content
Bead arrays
Cell Viability/Apoptosis
Sorting
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Cell sorting
FACSAria sorter:
Fixed nozzle/flow cell
High-speed sorting – 70,000 events/sec
3 lasers - 15 parameters
to achieve high purity, not higher than 10,000 events
the lower the frequency of the starting population the higher the chance for the low purity
take care of the necessary sorting time
keep cells on ice / medium
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Cell sorting – for validation
CD34 FITC-A
CD
146
APC
-A
100 101 102 103 104100
101
102
103
104
CD34 FITC-A
CD
146
APC
-A
100 101 102 103 104100
101
102
103
104
Morphology and vWF on FACS sorted CEC
Patient Healthy Donor
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Applications of Flow Cytometry Cel (subset) enumeration (e.g. Lymphocyte subsets, Stem cells) Celtyping using membrane / cytoplasmatic staining combinations
(e.g. leukemia / lymphoma typing / T-cell subsest) Cell cycle analysis using DNA content Bead arrays Cell Viability/Apoptosis Sorting Functional assays
intracellular pH intracellular calcium Phosporylated intracellular substrates / kinases oxidative burst phagocytosis tetramers
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Using Tetremers to identify CMV specific Cytotoxic T-lymphocyest
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Applications of Flow Cytometry Cel (subset) enumeration (e.g. Lymphocyte subsets, Stem cells) Celtyping using membrane / cytoplasmatic staining combinations
(e.g. leukemia / lymphoma typing / T-cell subsest) Cell cycle analysis using DNA content Bead arrays Cell Viability/Apoptosis Sorting Functional assays
intracellular pH intracellular calcium Phosporylated intracellular substrates / kinases oxidative burst phagocytosis tetramers Cytokine detection
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TH/C
Acquisition and analysis
(Kern et al. 1998 and 1999)
Picker 1997
Stimulation of PBMC with Peptides (1 nM / 1 ml / 106 Cells)
incubation (6-8 h) With Brefeldin A
fixation and permeabilisation
Staining
Laser 488 nm
Intracellular Cytokine Assay Method
(MHC-loading / Antigen presentation)
(Activation / Cytokine induction)
TH/C
TH/C Cytokine
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PBMC of a CMV+ patient stimulated with or without CMV lysate
Gated on CD3+ cells
CMV lysate: IFNg / CD3: 1,10 %
Control: IFNg / CD3: 0,01%
I.C. Cytokine Assay
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Combining advantages
R4R5
Tetramer + peptide
R4R5
Tetramer alone
no IFN-γ IFN-γ
Tet + Tet +
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Flow Cytometry PROS
Sensitive (one out of 104 –106) Capacity to analyse small subpopulations in a suspension/culture Combination of two or more assays in one tube Specificity Reproducible Objective Sorting capacity
CONS Need for skilled personnel Expensive (equipment) (Labour intensive)
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SOURCES
•Current Protocols in Cytometry: ISBN 0471 161 314, John Wiley & Sons, inc., New York
•Practical Flow Cytometry , 4th edition, Howard M. Shapiro: ISBN 0471 411 256, John Wiley & Sons, inc., New York