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Understanding Flow Cytometry

The Basic Concepts

Maree BagnaraProducts Sales Specialist/Account ManagerFlow Cytometry

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Successful Flow Cytometry data is driven by……..

● Understanding the Biology– Basic Immunology– Population(s) of interest– Antigen Expression

– Dim/bright– Ag proximity– Intracellular/extracellular

● Understanding the Technology– Fluorochrome Excitation / Emission spectra – Instrument Platform

– Lasers, Colour capability, Filters– Spectral Overlap - Compensation

● Reagent and Assay Optimization– Understanding of application methods

● GOOD SAMPLE PREPARATION

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● Direct Staining– Moab against target

species– Directly tagged with

Fluorochrome– Multi colour staining

Basic Immunology Fluorescence Techniques

Mouse anti–human CD4

Human CD4

Goat anti-mouse FITC

● Amplification of low density Ags

● Typically single colour

•Indirect Staining

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Fluorochrome Options for Gallios•Exercise 2

PI, 7AAD, PerCP, GFPsQDots, Hoechst

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Excitation/Emission of Common Fluorochromes

FITCFITC PEPE ECDECD PC5PC5 PECy5.5PECy5.5 PC7PC7

APCAPC Alexa 700Alexa 700 APCAlexa 700

APCAlexa 700

APC-H7APC-H7 APCAlexa 750

APCAlexa 750

Pacific BluePacific Blue Pacific OrangePacific Orange

525nm 575nm 620nm 675nm 700nm 770nm

450nm

675nm 700nm 700nm 750nm 750nm

488nm

647nm

405nm

•Krome Orange

•Sensitive to light

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• Current method – Signal:Noise

• Alternative Method – Stain Index (Sensitivity)

• Takes into account the spread of the background (negative population) vs positive population

• More “accurate” S:N• References

• Maecker et.al Cytometry 62A:169-173 (2004)• Hulspas et.al Cytometry 76B : 355-364 (2009)

•MFI(pos) / MFI(neg),

•MFI(pos) ‐MFI(neg)/ 2 x SD Background

Measuring “Brightness” Quantitation of Brightness over Background

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•Signal‐to‐Noise Ratio:•MFI(pos) / MFI(neg),

•420.15/0.37 = 1136

•Staining Index:•(MFI(pos)‐MFI(neg))/2*SD(neg),

•(420.15‐0.37)/2*0.23 = 913

•Information provided courtesy of Dr Michael Kapinsky, Beckman Coulter

Measuring “Brightness” Quantitation of Brightness over Background

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Compensation

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Compensation

•Exercise 2

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Tandem Dye TechnologySchematic of Energy Transfer Mechanism

Donor Molecule Acceptor Molecule

LASER

ECD 630 nmPC5 675 nmPC7 760 nmPerCPCy5.5 720 nmAPC Alexa 700 700nm760nm

PE (488) Texas RedPE (488) Cy5PE (488) Cy7PerCP (488) Cy5.5APC (635) Alexa700

Commercial Product

488 nmEmitted wavelength

Donor Molecule Acceptor MoleculeExcitation

PE Cy5

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Tandem Dye Technology

● Better sensitivity over synthetic dyes

● Not equivalent from manufacturer to manufacturer

● Issues

– Variation in energy transfer– Impacts compensation/instrument setup

– Photostability

– Stability

– Binding Specificity– Cy5 can bind nonspecifically to monocytes

– Lot to lot variability

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Same Tandem – Different Vendor

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Conjugate Choices

● Fluorochrome choice dependent on antigen density– Bright dye - low density antigens– Dimmer dye - high density antigens

● Critical for multi-color reagents– Proper compensation– Detection of dim/rare events – Dye conjugate interactions – do they “work” together?

– Non-specific binding– Steric Hindrance– Fluorescent quenching

High density Ag/Dim dye

Low density Ag/Bright dye

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PBPs, Tandems, or Synthetic Dyes?

● Cytoplasmic antigens:– Phycobiliproteins (PE, APC), Synthetic Dyes (Cyan and Alexa

Dyes)best– Tandem dyes can be degraded by cellular enzymes– Alexa Fluor 488 better than FITC – lower background

Intracellular Antigen DetectionIntracellular Antigen Detection

● Nuclear antigens:– Phycobiliproteins or tandem dyes: protein size may hinder binding– Close proximity can lead to energy transfer between dyes

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Compensation – Normal Human Lymphocytes

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Compensation – Normal Human Lymphocytes

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Understanding Colour CompensationBack to Basics Set-up for Compensation

● Neg controls to set PMT HV – Isotype matched and /or “blank” sample– Place negative cells in the first decade

● Single positive controls for each fluorochrome / CD8 of each flurochrom– Use biological sample or antibody capture beads such as VERSACOMP

● Verifier tube to check settings

● Set up a single protocol with histograms for all possible fluorochrome combinations

● Run each tube through protocol, making adjustments to compensation as required

● Final tube should be a “verifier’ which checks that the settings are correct

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Compensation Matrix

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Compensation Adjustment

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•S       I       L       E       N       T

•U  N  T  O  U  C  H  A  B  L  E

•U  N  T  O  U  C  H  A  B  L  E

•Classification is specific•for each combination of

•antibodies and conjugated•dyes on a given

•hardware configuration 

•Untouchable =•No overspill from•other dyes (clean row)

•Silent =•No overspill into•other channels (clean column)

•Information provided courtesy of Dr Michael Kapinsky, Beckman Coulter

Spillover Guide for the Gallios

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Multi-Color Combinations: What Can Go Wrong?

Size

+

++ +

+

++

+

+

+

+++

++

+

+++

Charge

++

+

ConcentrationProximity

Conjugate interactions can make the difference between right and wrong results •Decreased Mean Fluorescence or decreased % POS

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Non-Specific Binding of Dyes

● Root Cause– Low affinity binding of conjugate to

irrelevant populations – usually FcR

– Specific to cyanine dyes

– Other large molecule conjugates also display problem

● Blocking antibody - unlabelled– Same species as test Ab– Bind to FcR– Use Ig fraction rather than serum– 30ug Ig fraction per 106 cells in 100 ul.

Monocyte binding of Cyanine dye

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Optimising Moabs

● Perform titration curves for each conjugate by serial dilution assay– Plot Fluorescence Intensity vs dose for both positive and

negative signal – Choose optimal dose

– Saturation area of curve– Highest Signal to Noise

● Prepare combination for Moab Cocktails– Matrix Titre

● Evaluate for– Non-specific binding– Steric Hindrance– Fluorescence Quenching

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Dose Titration

● Perform titration curves for each conjugate by serial dilution assay

MoAb conjugate

Dilu

tion

● Plot Fluorescence intensity vs dose for both positive signal and noise

● Determine Signal/Noise Ratio

● Choose optimal conc range (2-3 points)

– Saturation area of curve

– Highest S/N

NEAT

1:21:41:8

Mea

n C

hann

el

0

5

10

15

20

25

30

35

0 0.5 1 1.5 2

Ab conc./test (μg) / Dilution

positive pop

negative pop

Mea

n C

han

nel

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Moab Cocktail Optimisation

● Use titration information for Moab cocktail matrix– CDx at three doses – 1,2,3– CDy at three doses – a,b,c– Ideal [Ab] 0.01-1.0 ug/test staining 1X106 cells in 100ul– Determine saturation point– Note: each Moab volume dilutes the other!

● Evaluate performance for major interactions– Non-specific binding– Steric Hindrance– Fluorescence Quenching– Verify on multiple samples

N 1:2 1:4

x

y

N

1:2

1:4

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Optimal Formulation

● Formulation Choice– Choose highest Signal/Noise within saturation area of curve– Moab mix must demonstrate equivalent performance to single

color reagent controls– No evidence of any interactions

0.0

5.0

10.0

15.0

20.0

0 0.2 0.4 0.6 0.8 1

Dose (ug/test)

Fluo

resc

ence

nce

S/NPositive MFINegative MFI

Single CD4 FITC CD4 FITC (with CD8 PE)

•0.0

•5.0

•10.0

•15.0

•20.0

•0 •0.2 •0.4 •0.6 •0.8 •1

•Dose (ug/test)

•Flu

ores

cenc

ence

•S/N•Positive MFI•Negative MFI

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Specimen Processing

● Sample presentation– Anticoagulants – ETDA, Heparin– Cell Lines – Adherent, Non-adherent– Cell Media – Phenol Red– Clumping – Flow Cytometer nightmare!

● Age of sample– EDTA – 8 – 30 hours– Gate out dead cells– Dye for Viability – PI, 7AAD

● Red Cell Contamination– Many RBC Lysis Products

– Optilyse– Immunoprep– Ammonium Chloride

– Ficoll Hyapaque

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Sample Preparation

● Pre and Post Wash– Remove interfering dyes from Culture medium– Post wash can improve signal to noise– Loss of Cells

● Washing Medium– Check pH of PBS – pH7.2– FCS ? B etter than BSA?– Check pH after adding buffering serum

● Autofluorescence– Gate autofluorescence population and backgate onto

Scatter plot – ensure cells of interest are not contained

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Sample Preparation

● Antibody-Antigen binding is a dynamic process– Influenced by three parameters

– Time– 1min – 15mins– Size of fluorochrome, cocktail size, Ag density

– Temperature– Room Temperature. If delay - refrigerate

– Concentration of Moab– Use in excess rather than less– Are you using adequate Moab for the “worst" case scenario– Questionable data?

» Use it at manufacturer’s recommendation on a normal specimen.» Run Moabs as a SINGLE stain if using in a cocktail

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Inhouse Cocktails!

● Do not store pre-mixes for extended periods of time– Do NOT dilute / pre-mix Moab in original vial– Do a time course study– Add protein

● Dark vials

● When in doubt – run a normal control specimen!

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Negative Controls – what do they do?

● Fluorescence due to non-specific binding of MoAb to cell– Mainly due to binding to Fc receptors on the cell surface– Dependant on IgG subclass

– IgG1<IgG2a~IgG2b<IgM (Most “sticky”)

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Why Use Negative Controls

● Set fluorescence PMT HV to obtain best signal to noise ratio– Usually in the first decade– Set Analysis regions

0.1-2%

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Negative Control

● A non-specific MoAb of the same IgG subclass as the specific MoAb used to assess positive staining

● Should be at the same protein concentration as the MoAb

● Should have the same Fluorchrome/Protein ratio as the specific MoAb

● More common to use the cells that stain negative for the moab of interest – “internal Control”

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Where do we put the region?

Negative

R1

Positive

Fluorescence

R1

R1

45%

38%

2%•Often needs to be moved with test samples

•Subjective.

•Move the region if necessary!

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Where do we put the region?

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Low Ag density on cells

● How do we handle this sample?– 30% pos or 100% weak pos?

● Cells that are negative for an Ag may be more useful than a neg control

(Log) Fluorescence

Negative control

Specific stain

R1

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Application Standardisation

● Standardise the fluorescent light emitting from the Photo Multiplier Tubes

● Enables data comparison between samples and across instruments

● Maintain compensation settings

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PMT HV = 456 V

DAY 0

DAY 24DAY 24 - Drug XYZ

HV to 350 V

Mean Channel Standardisation-Using FlowSet beads

Decrease PMT HV to 350 V

DAY 24PMT HV = 456 V

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Standards In Cytometry