Steps to Success with
Multicolor Flow Cytometry
Holden T. Maecker
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
Outline
1. Configure your instrument Number and type of lasers Number of PMTs per laser Choice of filters and dichroic mirrors
These choices will determine: What fluorochromes you can use effectively How well certain fluorochrome combinations
will perform
Holden Maecker, Flow Cytometry Consulting
How do we measure performance?
W2
W1
D
Where D = difference between positive and negative peak medians, andW = 2 x rSD (robust standard deviation)
Stain Index = D / W
Resolution Sensitivity:
Holden Maecker, Flow Cytometry Consulting
CD127 PE
300 400 500 600 7000
5
10
15
20
25
30
35
40
25 mW green laser (532 nm)
100 mW blue laser (488 nm)
25 mW blue laser (488 nm)
PMT voltage
Sta
in i
nd
ex
An Example: Green vs. Blue Lasers
Green laser more efficient for PE and PE tandems Green laser less efficient for FITC, PerCP and GFP
Holden Maecker, Flow Cytometry Consulting
Second Example: Filters and Spillover
Holden Maecker, Flow Cytometry Consulting
Outline
2. Characterize your instrument Obtain minimum baseline PMT settings Track performance over time
This will allow you to: Run the instrument where it is most sensitive Be alert to changes in the instrument that
might affect performance
Holden Maecker, Flow Cytometry Consulting
SDEN
Baseline PMTV is set by placing the dim bead MFI to equal 10X SDBaseline PMTV is set by placing the dim bead MFI to equal 10X SDENEN
460 V
Automated baseline PMT voltage determination in Diva 6.0
Holden Maecker, Flow Cytometry Consulting
Performance Tracking
A variety of parameters can be tracked: Linearity, CVs, laser alignment PMT voltages required to hit target values
Data can be visualized in Levey-Jennings plots:
400
425
450
475
500
525
550
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
PM
T V
olt
ag
e
FITC Channel (Blue laser)FITC Channel (Blue laser)
Holden Maecker, Flow Cytometry Consulting
Outline
3. Design your panel 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: Maintain resolution sensitivity where you
most need it Avoid artifacts of tandem dye degradation
Holden Maecker, Flow Cytometry Consulting
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
Holden Maecker, Flow Cytometry Consulting
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
Compensation requirements for tandem dye conjugates can vary, even between two experiments with the same antibody Degrade with exposure to light, temperature, and
fixation Stained cells are most vulnerable
Solutions: Minimize exposure to above agents Use BD stabilizing fixative if a final fix is necessary Run experiment-specific compensation
Holden Maecker, Flow Cytometry Consulting
False positives due to tandem degradation
A.
False positives inAPC channel reducedin absence of APC-Cy7
False positivesin PE channelremain
Gating scheme CD8 APC-Cy7+ cells CD4 PE-Cy7+ cells
B.
With CD8 APC-Cy7 and CD4 PE-Cy7:
Without CD8 APC-Cy7:
Holden Maecker, Flow Cytometry Consulting
New tandems can be more stable
APC-H7 as a replacement for APC-Cy7:
Comparison of Sample Stability
(in BD Stabilizing Fixative at RT)
0
50
100
150
200
250
0 1 2 4 6 8 24 48
Hours of light exposure
% S
pillo
ver
CD4 APC-H7
CD8 APC-H7
CD4 APC-Cy7
CD8 APC-Cy7
Holden Maecker, Flow Cytometry Consulting
Antibody titration basics
For most purposes, the main objective is to maximize signal:noise (pos/neg separation) This may occur at less than saturated staining This may or may not be the manufacturer’s
recommended titer Titer is affected by:
Staining volume (e.g., 100 L) Number of cells (not critical up to ~5x106) Staining time and temperature (e.g., 30 min RT) Type of sample (whole blood, PBMC, etc.)
Holden Maecker, Flow Cytometry Consulting
Antibody titration example
11010010001
10
100
1000
10000 signal
noise
S:N
ng antibody
Holden Maecker, Flow Cytometry Consulting
Outline
4. Optimize settings for your panel Derive experiment-specific PMT settings Run compensation controls for each
experiment
This will allow you to: 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:• 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
Holden Maecker, Flow Cytometry Consulting
Experiment-specific setup for existing panel
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 Instrument setup controls (e.g., CompBeads) Gating controls (e.g., FMO) Biological controls (e.g., unstimulated
samples, healthy donors)
This will allow you to: 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
Do I need to use the same antibody for compensation as I use in the experiment?
Yes, for certain tandem dyes (e.g., PE-Cy7, APC-Cy7)
Are capture beads better than cells for compensation?
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)?
Yes, although with optimal fix/perm protocols this may make little difference
Holden Maecker, Flow Cytometry Consulting
Comparison of gating controls
Holden Maecker, Flow Cytometry Consulting
Consider using lyophilized reagents
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 Visually inspect compensation Visually inspect gating Set sample acceptance criteria
This will allow you to: Avoid classification errors and false
conclusions due to improper compensation and/or gating, or sample artifacts
Holden Maecker, Flow Cytometry Consulting
Visually inspect compensation
Create a template containing dot plots of each color combination in your experiment, then examine a fully stained sample for possible compensation problems
Yikes!
Holden Maecker, Flow Cytometry Consulting
Visually inspect gating
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
IL-2 PE
IFN
FIT
C
Holden Maecker, Flow Cytometry Consulting
Types of sample acceptance criteria
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
Maecker, H. T., Frey, T., Nomura, L. E., and Trotter, J. 2004. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62: 169.
Maecker, H. T., and Trotter, J. 2006. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69: 1037.
Roederer, M. 2008. How many events is enough? Are you positive? Cytometry A 73: 384-385.
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
Laurel Nomura Margaret Inokuma Maria Suni Maria Jaimes Smita Ghanekar Jack Dunne Skip Maino
Joe Trotter Dennis Sasaki Marina Gever
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