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 or 2o fluorescence

Sort single particles for subsequent analysis

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

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) on 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

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)

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-C

D3

inte

nsity

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

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

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

R4R5

Tetramer + peptide

R4R5

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