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 10 5 to 10 6 particles in less than 1 min  Measure particle-scatter as well as innate fluorescence or 2 o fluorescence  Sort single particles for subsequent analysis

Flow Cytometry Publications/year 27042 30000 21944 25000 15180 apers 20000 Paper 12310 15000 6652 10000 2697 25 0 5000 0 1975 1980 1985 1990 1995 2000 2005 2010 YEAR YEARS Data taken from Medline search using the keywords: “Flow Cytometry”

FLUIDICS Getting the cells in the right place (at the rigth time) using hydrodynamic focusing 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. J.Paul Robinson http://www.cyto.purdue.edu

PMT 5 PMT 4 Sample PMT 3 Dichroic Filters Flow cell PMT 2 Scatter PMT 1 Laser Sensor Bandpass Filters

Optical Filters Dichroic Filter/Mirror at 45 deg Light Source Transmitted Light Reflected light J.Paul Robinson http://www.cyto.purdue.edu

Long and short Pass Filters 520 nm Long Pass Filter Light Source Transmitted Light >520 nm Light 575 nm Short Pass Filter Light Source Transmitted Light <575 nm J.Paul Robinson Light 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 Fluidics  Flow in single-file through  An illuminated volume where they  Scatter light and emit fluorescence Optics  That is collected, filtered and  Converted to digital values Electronics  that are strored on a computer

Data analysis - 1-parameter histogram Positive Negative Brighter Count Dimmer 6 4 1 1 2 3 4 6 7 150 160 170 .. 190 Channel Number Fluorescence picked up from the FITC PMT

Data analysis - 2-parameter histogram or dotplot Single Double Positive Positive PE Population Population PE FL Negative Population Single Positive FITC FL FITC Population

Light Scatter properties (1)

Light Scatter properties (2)

Light Scatter properties (3)

Light Scatter properties (4)

Scatter properties (3) 1000 800 Neutrophils Side Scatter 600 400 Monocytes 200 Lymphocytes 0 0 200 400 600 800 1000 Forward Scatter Platelets

Fluorescence Incident Emitted Fluorescent Light Energy Light Energy Fluorescein λ = 488 nm λ ≅ 530 nm Molecule HO O C CO 2 H Antibody

FITC spectral characteristics FITC PMT BAND PASS

A TWO COLOR OPTICAL BENCH FITC PMT PE PMT

Spectral overlap in PE channel FITC PMT PE PMT BAND PASS BAND PASS SPILLOVER

PE spectral characteristics FITC PMT PE PMT BAND PASS BAND PASS SPILLOVER

INTRA-LASER SPILLOVER the fluorochrome emission is mainly skewed towards the right PE PE-TR PE-CY5.5 PE-CY7 FITC PMT PMT PMT PMT PMT FITC EMISSION

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 10 6 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 Surface and Intracellular staining  100µL - 10 6 cells + 10µL mAb(s)  Perform cell surface staining  Incubate for 15’ at RT in the dark  Fix cells in (1%PFA)  Wash with 2 mL assaybuffer  Wash with 2 mL assaybuffer  Centrifuge 10’ 500 g  Centrifuge 10’ 500 g  Fix cells in 1ml PBS/1%PFA  Permeabilize Cells (Triton/saponin)  Acquire on FCM  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 PE-CD3 intensity unstained 6 ng/ml 60 ng/ml 300 ng/ml 600 ng/ml Side Light Scatter  Typical manufacturer’s recommendations: X µ L per 1E6 cells (in 0.5 ml).  Background increases with increasing number of Ab molecules.

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 4 10 3 CD146 APC-A 10 2 10 1 Patient 10 0 10 0 1 2 3 4 10 10 10 10 10 CD34 FITC-A 4 10 3 CD146 APC-A 10 2 10 1 10 Healthy 0 10 Donor 0 1 2 3 4 10 10 10 10 10 Morphology and vWF on FACS sorted CEC CD34 FITC-A

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

Stimulation of PBMC with Intracellular Cytokine Peptides (1 nM / 1 ml / 10 6 Cells) Assay Method (MHC-loading / Antigen presentation) incubation (6-8 h) With Brefeldin A (Activation / Cytokine induction) T H/C fixation and Picker 1997 permeabilisation T H/C Cytokine Staining Laser T H/C 488 nm Acquisition and analysis (Kern et al. 1998 and 1999)

I.C. Cytokine Assay PBMC of a CMV+ patient stimulated with or without CMV lysate Gated on CD3+ cells Control: CMV lysate: IFNg / CD3: 0,01% IFNg / CD3: 1,10 %

Combining advantages Tetramer alone Tetramer + peptide IFN- γ no IFN- γ Tet + Tet + R5 R4 R5 R4

Flow Cytometry PROS  Sensitive (one out of 10 4 –10 6 )  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)