Understanding Flow Cytometry
The Basic Concepts
Maree Bagnara
Products Sales Specialist/Account Manager
Flow Cytometry
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Successful Flow Cytometry data is driven by……..
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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
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GOOD SAMPLE PREPARATION
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Basic Immunology Fluorescence Techniques
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Direct Staining
– Moab against target
species
– Directly tagged with
Fluorochrome
– Multi colour staining
•Indirect Staining
Mouse anti–human CD4
Goat anti-mouse FITC
Human CD4
●
Amplification of low density
Ags
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● Typically single colour
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Fluorochrome Options for Gallios
•Exercise 2
QDots, Hoechst
PI, 7AAD, PerCP, GFPs
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Excitation/Emission of Common Fluorochromes
525nm
575nm
FITC
620nm
PE
675nm 700nm
ECD
PC5
770nm
PECy5.5
PC7
488nm
675nm
700nm
APC
Alexa 700
700nm
APCAlexa
700
750nm
APC-H7
750nm
APCAlexa
750
647nm
Pacific Blue
Pacific
Orange
•Krome
Orange
450nm
405nm
•Sensitive to light
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Measuring “Brightness” Quantitation of Brightness over
Background
•
Current method – Signal:Noise
•MFI(pos) / MFI(neg),
•
Alternative Method – Stain Index
(Sensitivity)
•MFI(pos) ‐ MFI(neg)/ 2 x SD Background
•
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)
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Measuring “Brightness” Quantitation of Brightness over
Background
•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
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•Information provided courtesy of Dr Michael Kapinsky, Beckman Coulter
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Compensation
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Compensation
•Exercise 2
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Tandem Dye Technology
Schematic of Energy Transfer Mechanism
Donor Molecule
Excitation
Acceptor Molecule
Emitted wavelength
488 nm
LASER
PE
Donor Molecule
PE
PE
PE
PerCP
APC
Cy5
Acceptor Molecule
(488)
(488)
(488)
(488)
(635)
Texas Red
Cy5
Cy7
Cy5.5
Alexa700
Commercial Product
ECD
PC5
PC7
PerCPCy5.5 720 nm
APC Alexa 700
760nm
630 nm
675 nm
760 nm
700nm
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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
●
High density Ag/
Dim dye
Fluorochrome choice dependent on antigen density
– Bright dye - low density antigens
– Dimmer dye - high density antigens
Low density Ag/
Bright dye
●
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
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PBPs, Tandems, or Synthetic Dyes?
Intracellular Antigen Detection
●
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
●
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 Compensation
Back 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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Spillover Guide for the Gallios
•Untouchable =
•No overspill from
•other dyes (clean row)
•U N T O U C H A B L E
•S
I
L
E
N
T
•U N T O U C H A B L E
•Silent =
•No overspill into
•other channels (clean column)
•Classification is specific
•for each combination of
•antibodies and conjugated
•dyes on a given
•hardware configuration
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•Information provided courtesy of Dr Michael Kapinsky, Beckman Coulter
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Multi-Color Combinations: What Can Go Wrong?
+
+
Size
+
Charge
+ +
+
+
+
+
+
+ ++ +
+
+ ++ +
+
+ +
Proximity
Concentration
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
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Root Cause
– Low affinity binding of conjugate to
irrelevant populations – usually FcR
Monocyte
binding of
Cyanine dye
– 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.
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Optimising Moabs
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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
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Evaluate for
– Non-specific binding
– Steric Hindrance
– Fluorescence Quenching
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Dilution
Dose Titration
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MoAb conjugate
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30
positive pop
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1:8
1:4
1:2
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Mean Channel
Mean Channel
Perform titration curves for
each conjugate by serial
dilution assay
NEAT
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10
●
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
negative pop
5
0
0
0.5
1
1.5
Ab conc./test (μg) / Dilution
2
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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
x
N 1:2 1:4
●
N 1:2 1:4
y
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Optimal Formulation
Single CD4 FITC
CD4 FITC (with CD8 PE)
•20.0
15.0
•Fluorescencence
Fluorescencence
20.0
S/N
Positive MFI
Negative MFI
10.0
5.0
0.0
0
0.2
0.4
0.6
Dose (ug/test)
●
0.8
1
•15.0
•S/N
•Positive MFI
•Negative MFI
•10.0
•5.0
•0.0
•0
•0.2
•0.4
•0.6
•0.8
•1
•Dose (ug/test)
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
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Specimen Processing
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Sample presentation
–
–
–
–
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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
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Red Cell Contamination
– Many RBC Lysis Products
– Optilyse
– Immunoprep
– Ammonium Chloride
– Ficoll Hyapaque
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Sample Preparation
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Pre and Post Wash
– Remove interfering dyes from Culture medium
– Post wash can improve signal to noise
– Loss of Cells
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Washing Medium
– Check pH of PBS – pH7.2
– FCS ? B
etter than BSA?
– Check pH after adding buffering serum
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Autofluorescence
– Gate autofluorescence population and backgate onto
Scatter plot – ensure cells of interest are not contained
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Sample Preparation
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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!
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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?
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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
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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
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A non-specific MoAb of the same IgG subclass as the specific
MoAb used to assess positive staining
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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
2%
•Often needs to be
moved with test
samples
R
1
Positive
45%
R
1
•Subjective.
•Move the region if
necessary!
38%
R
1
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Fluorescence
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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
Negative control
Specific stain
R1
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(Log) Fluorescence
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Application Standardisation
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Standardise the fluorescent light emitting from the
Photo Multiplier Tubes
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Enables data comparison between samples and
across instruments
●
Maintain compensation settings
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Mean Channel Standardisation-Using FlowSet beads
DAY 24
PMT HV = 456 V
PMT HV = 456 V
DAY 0
DAY 24 - Drug XYZ
DAY 24
HV to 350 V
Decrease
PMT HV to
350 V
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Standards In Cytometry
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