Transcript No Slide Title

Steps to Success with
Multicolor Flow Cytometry
Holden T. Maecker
Outline
1. Configure your instrument
2. Characterize your instrument
3. Design your panel
4. Optimize settings for your panel
5. Run appropriate controls
6. QC your data
Holden Maecker, Flow Cytometry Consulting
Outline
1. Configure your instrument
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Number and type of lasers
Number of PMTs per laser
Choice of filters and dichroic mirrors
These choices will determine:
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What fluorochromes you can use effectively
How well certain fluorochrome combinations
will perform
Holden Maecker, Flow Cytometry Consulting
How do we measure performance?
Resolution Sensitivity:
D
W1
W2
Stain Index = D / W
Where
D = difference between positive and negative peak medians, and
W = 2 x rSD (robust standard deviation)
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An Example: Green vs. Blue Lasers
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Green laser more efficient for PE and PE tandems
Green laser less efficient for FITC, PerCP and GFP
CD127 PE
40
35
Stain index
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30
25 mW green laser (532 nm)
25
100 mW blue laser (488 nm)
20
25 mW blue laser (488 nm)
15
10
5
0
300 400 500 600 700
PMT voltage
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Second Example: Filters and Spillover
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Outline
2. Characterize your instrument
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Obtain minimum baseline PMT settings
Track performance over time
This will allow you to:
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Run the instrument where it is most sensitive
Be alert to changes in the instrument that
might affect performance
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Automated baseline PMT voltage
determination in Diva 6.0
Baseline PMTV is set by placing the dim bead MFI to equal 10X SDEN
460 V
SDEN
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Performance Tracking
A variety of parameters can be tracked:
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Linearity, CVs, laser alignment
PMT voltages required to hit target values
Data can be visualized in Levey-Jennings
plots:
550
PMT Voltage
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FITC Channel (Blue laser)
525
500
475
450
425
400
10/22/04 11/11/04 12/01/04 12/21/04 01/10/05 01/30/05 02/19/05 03/11/05
Time
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Outline
3. Design your panel
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Reserve brightest fluorochromes for
dimmest markers and vice versa
Avoid spillover from bright populations into
detectors requiring high sensitivity
Beware of tandem dye issues
Titrate antibodies for best separation
This will allow you to:
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Maintain resolution sensitivity where you
most need it
Avoid artifacts of tandem dye degradation
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Various fluorochromes-stain index
Reagent
Clone
Filter
Stain Index
PE
RPA-T4
585/40
356.3
Alexa 647
RPA-T4
660/20
313.1
APC
RPA-T4
660/20
279.2
PE-Cy7
RPA-T4
780/60
278.5
PE-Cy5
RPA-T4
695/40
222.1
PerCP-Cy5.5
Leu-3a
695/40
92.7
PE-Alexa 610
RPA-T4
610/20
80.4
Alexa 488
RPA-T4
530/30
75.4
FITC
RPA-T4
530/30
68.9
PerCP
Leu-3a
695/40
64.4
APC-Cy7
RPA-T4
7801/60
42.2
Alexa 700
RPA-T4
720/45
39.9
Pacific Blue
RPA-T4
440/40
22.5
AmCyan
RPA-T4
525/50
20.2
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Spillover affects resolution sensitivity
Without CD45 AmCyan:
With CD45 AmCyan:
CD19 FITC
Note that this is only an issue when the two markers (CD45
and CD19) are co-expressed on the same cell population.
Holden Maecker, Flow Cytometry Consulting
Special requirements of tandem dyes
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Compensation requirements for tandem dye
conjugates can vary, even between two
experiments with the same antibody
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Degrade with exposure to light, temperature, and
fixation
Stained cells are most vulnerable
Solutions:
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Minimize exposure to above agents
Use BD stabilizing fixative if a final fix is necessary
Run experiment-specific compensation
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False positives due to tandem degradation
A. With CD8 APC-Cy7 and CD4 PE-Cy7:
Gating scheme
B. Without CD8 APC-Cy7:
CD8 APC-Cy7+ cells
CD4 PE-Cy7+ cells
False positives in
APC channel reduced
in absence of APC-Cy7
False positives
in PE channel
remain
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New tandems can be more stable
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APC-H7 as a replacement for APC-Cy7:
Comparison of Sample Stability
(in BD Stabilizing Fixative at RT)
250
% Spillover
200
CD4 APC-Cy7
150
CD8 APC-Cy7
CD4 APC-H7
100
CD8 APC-H7
50
0
0
1
2
4
6
8
Hours of light exposure
24
48
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Antibody titration basics
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For most purposes, the main objective is to
maximize signal:noise (pos/neg separation)
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This may occur at less than saturated staining
This may or may not be the manufacturer’s
recommended titer
Titer is affected by:
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Staining volume (e.g., 100 mL)
Number of cells (not critical up to ~5x106)
Staining time and temperature (e.g., 30 min RT)
Type of sample (whole blood, PBMC, etc.)
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Antibody titration example
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Outline
4. Optimize settings for your panel
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Derive experiment-specific PMT settings
Run compensation controls for each
experiment
This will allow you to:
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Use settings most appropriate for your panel
Avoid gross errors of compensation
Holden Maecker, Flow Cytometry Consulting
Experiment-specific setup for a new panel
1. Set voltages to achieve baseline target values
2. Run single-stained CompBeads to see if each bead is at least
2x brighter in its primary detector vs. other detectors
•
If not, increase voltage in the primary detector (beware: potential
reagent problem)
3. Run fully-stained cells and:
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Decrease voltages for any detectors where events are off-scale
Increase voltages for any detectors where low-end resolution is
poor (theoretically should not be necessary)
4. Re-run single-stained CompBeads and calculate compensation
5. Re-run fully-stained cells and repeat step 3 (if further changes
made, re-run compensation)
6. Save experiment-specific settings as target values
7. Run samples
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Experiment-specific setup for existing panel
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Set voltages to achieve experiment-specific target
channels
Run single-stained CompBeads and calculate
compensation
Run samples
Holden Maecker, Flow Cytometry Consulting
Outline
5. Run appropriate controls
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Instrument setup controls (e.g., CompBeads)
Gating controls (e.g., FMO)
Biological controls (e.g., unstimulated
samples, healthy donors)
This will allow you to:
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Obtain consistent setup and compensation
Gate problem markers reproducibly
Make appropriate biological comparisons
and conclusions
Holden Maecker, Flow Cytometry Consulting
CompBeads as single-color controls
CompBeads provide a convenient
way to create single-color
compensation controls:
• Using the same Abs as in the
experimental samples
• Creating a (usually) bright and
uniform positive fluorescent peak
• Without using additional cells
Holden Maecker, Flow Cytometry Consulting
Frequent compensation questions
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Do I need to use the same antibody for
compensation as I use in the experiment?
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Are capture beads better than cells for
compensation?
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Yes, for certain tandem dyes (e.g., PE-Cy7, APC-Cy7)
Usually, as long as the antibody binds to the bead and is
as bright or brighter than stained cells
Should compensation controls be treated the same
as experimental samples (e.g., fixed and
permeabilized)?
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Yes, although with optimal fix/perm protocols this may
make little difference
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Comparison of gating controls
Holden Maecker, Flow Cytometry Consulting
Consider using lyophilized reagents
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Lyophilization provides increased stability,
even at room temperature or 37oC
One batch of reagents can be used for an
entire longitudinal study
Pre-configured plates can avoid errors of
reagent addition
Complex experiments (multiple stimuli,
multiple polychromatic staining cocktails)
become easier
Lyophilized cell controls can provide run-to-run
standardization
Holden Maecker, Flow Cytometry Consulting
Outline
6. QC your data
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Visually inspect compensation
Visually inspect gating
Set sample acceptance criteria
This will allow you to:
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Avoid classification errors and false
conclusions due to improper compensation
and/or gating, or sample artifacts
Holden Maecker, Flow Cytometry Consulting
Visually inspect compensation
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Create a template containing dot plots of each
color combination in your experiment, then
examine a fully stained sample for possible
compensation problems
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Yikes!
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Visually inspect gating
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Check gating across all samples in the
experiment
Gates may need to be adjusted across donors
and/or experimental runs; dynamic (e.g.,
snap-to) gates may help in some cases
IFNg FITC
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IL-2 PE
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Types of sample acceptance criteria
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Minimum viability and recovery for
cryopreserved PBMC
Minimum number of events collected in an
appropriate gate (e.g., lymphocytes)
Minimum number of events within a region
of interest, to calculate an accurate
percentage
Holden Maecker, Flow Cytometry Consulting
Outline
1. Configure your instrument
2. Characterize your instrument
3. Design your panel
4. Optimize settings for your panel
5. Run appropriate controls
6. QC your data
Holden Maecker, Flow Cytometry Consulting
A question for you to answer
How many colors can you combine and still
have robust results? This depends on:
-The experimental question
-The instrument used
-The markers to be combined
Holden Maecker, Flow Cytometry Consulting
References
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Maecker, H. T., Frey, T., Nomura, L. E., and Trotter, J. 2004.
Selecting fluorochrome conjugates for maximum sensitivity.
Cytometry A 62: 169.
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Maecker, H. T., and Trotter, J. 2006. Flow cytometry controls,
instrument setup, and the determination of positivity. Cytometry A 69:
1037.
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Roederer, M. 2008. How many events is enough? Are you positive?
Cytometry A 73: 384-385.
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McLaughlin, B. E., N. Baumgarth, M. Bigos, M. Roederer, S. C. De
Rosa, J. D. Altman, D. F. Nixon, J. Ottinger, C. Oxford, T. G. Evans,
and D. M. Asmuth. 2008. Nine-color flow cytometry for accurate
measurement of T cell subsets and cytokine responses. Part I: Panel
design by an empiric approach. Cytometry A 73: 400-410.
Holden Maecker, Flow Cytometry Consulting
Acknowledgements
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Laurel Nomura
Margaret Inokuma
Maria Suni
Maria Jaimes
Smita Ghanekar
Jack Dunne
Skip Maino
 Joe Trotter
 Dennis Sasaki
 Marina Gever
Holden Maecker, Flow Cytometry Consulting