FLOW CYTOMETRY November 1th, 2018 Jaco Kraan, PhD Dept. of - - PowerPoint PPT Presentation

Molecular Medicine Postgraduate School Course Biomedical Research Techniques

FLOW CYTOMETRY

November 1th, 2018

Jaco Kraan, PhD

• Dept. of Medical Oncology

Erasmus MC Cancer Institute j.kraan@erasmusmc.nl

FLOW CYTOMETRY

• Introduction
• Principle of the instrument
• Fluidics
• Optics
• Electronics
• Analysis of results
• Applications on a flowcytometer
• Examples and results
• Pros and cons

Flow Cytometers

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
• r 2o fluorescence
• Sort single particles for subsequent analysis

Flow Cytometry Publications/year

YEARS

Paper apers

Data taken from Medline search using the keywords: “Flow Cytometry”

5000 10000 15000 20000 25000 30000 1975 1980 1985 1990 1995 2000 2005 2010 25 2697 6652 12310 15180 21944 27042

YEAR

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.

PMT 1 PMT 2 PMT 5 PMT 4

Dichroic Filters Bandpass Filters

Laser

Flow cell

PMT 3

Scatter Sensor

Sample

Optical Filters

Dichroic Filter/Mirror at 45 deg

Reflected light Transmitted Light Light Source

J.Paul Robinson http://www.cyto.purdue.edu

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

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)

• n a 1 Parameter Histogram or 2 Parameter Histogram.
• All events are individually correlated for all the parameters

collected

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

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

FITC FL PE FL

Negative Population Single Positive FITC Population Single Positive PE Population Double Positive Population

Data analysis - 2-parameter histogram or dotplot

Light Scatter properties (1)

Light Scatter properties (2)

Light Scatter properties (3)

Light Scatter properties (4)

Side Scatter

200 400 600 800 1000 200 400 600 800 1000 Lymphocytes Monocytes Neutrophils Platelets

Forward Scatter

Scatter properties (3)

Fluorescence

λ = 488 nm

Emitted Fluorescent Light Energy Antibody Incident Light Energy Fluorescein Molecule

λ ≅ 530 nm HO CO2H O C

FITC spectral characteristics

FITC PMT BAND PASS

A TWO COLOR OPTICAL BENCH

FITC PMT PE PMT

Spectral overlap in PE channel

FITC PMT BAND PASS PE PMT BAND PASS SPILLOVER

PE spectral characteristics

FITC PMT BAND PASS PE PMT BAND PASS SPILLOVER

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

INTRA-LASER SPILLOVER

Setting electronic compensation for spectral overlap ('color compensation')

• Using single labeled control cells or beads

Setting electronic compensation for spectral overlap ('color compensation')

• Validate using multiple labelled control cells

Setting electronic compensation for spectral overlap ('color compensation')

• UNCOMPENSATED !
• multiple labeled lymphocytes

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.

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

Fluorochrome and mAb selection considerations

• Titration of antibodies – to reduce non-specific mAb binding

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-CD3 intensity

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

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)

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

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

Cell sorting – for validation

CD34 FITC-A CD146 APC-A 10 10

1

10

2

10

3

10

4

10 10

1

10

2

10

3

10

4

CD34 FITC-A CD146 APC-A 10 10

1

10

2

10

3

10

4

10 10

1

10

2

10

3

10

4

Morphology and vWF on FACS sorted CEC

Patient Healthy Donor

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

Using Tetremers to identify CMV specific Cytotoxic T-lymphocyest

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

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

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

Combining advantages

R4 R5

Tetramer + peptide

R4 R5

Tetramer alone

no IFN-γ IFN-γ Tet + Tet +

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)

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