1. Steps to Success with Multicolor Flow Cytometry
Holden T. Maecker

2. Outline Configure your instrument Characterize your instrument
Design your panel
Optimize settings for your panel
Run appropriate controls
QC your data

3. Outline Configure your instrument These choices will determine:
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

4. How do we measure performance?
Resolution Sensitivity: D W2 W1
Stain Index = D / W
Where D = difference between positive and negative peak medians, and
W = 2 x rSD (robust standard deviation)

5. An Example: Green vs. Blue Lasers
Green laser more efficient for PE and PE tandems
Green laser less efficient for FITC, PerCP and GFP
CD127 PE
300
400
500
600
700 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
Stain index

6. Second Example: Filters and Spillover

7. Outline Characterize your instrument This will allow you to:
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

8. 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

9. 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:
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425
450
475
500
525
550
10/22/04
11/11/04
12/01/04
12/21/04
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01/30/05
02/19/05
03/11/05
Time
PMT Voltage
FITC Channel (Blue laser)

10. Outline Design your panel This will allow you to:
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

11. Various fluorochromes-stain index
Reagent
Clone
Filter
Stain Index PE
RPA-T4
585/40
356.3
Alexa 647
660/20
313.1
APC
279.2
PE-Cy7
780/60
278.5
PE-Cy5
695/40
222.1
PerCP-Cy5.5
Leu-3a
92.7
PE-Alexa 610
610/20
80.4
Alexa 488
530/30
75.4
FITC
68.9
PerCP
64.4
APC-Cy7
7801/60
42.2
Alexa 700
720/45
39.9
Pacific Blue
440/40
22.5
AmCyan
525/50
20.2

12. 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.

13. 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

14. False positives due to tandem degradation
With CD8 APC-Cy7 and CD4 PE-Cy7:
Gating scheme
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 B.
Without CD8 APC-Cy7:

15. New tandems can be more stable
APC-H7 as a replacement for APC-Cy7:
Comparison of Sample Stability
(in BD Stabilizing Fixative at RT)
250
200
CD4 APC-Cy7
150
CD8 APC-Cy7
% Spillover
CD4 APC-H7
100
CD8 APC-H7 50 1 2 4 6 8 24 48
Hours of light exposure

16. 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 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.)

17. Antibody titration example

18. Outline Optimize settings for your panel This will allow you to:
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

19. Experiment-specific setup for a new panel
Set voltages to achieve baseline target values
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)
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)
Re-run single-stained CompBeads and calculate compensation
Re-run fully-stained cells and repeat step 3 (if further changes made, re-run compensation)
Save experiment-specific settings as target values
Run samples

20. Experiment-specific setup for existing panel
Set voltages to achieve experiment-specific target channels
Run single-stained CompBeads and calculate compensation
Run samples

21. Outline Run appropriate controls This will allow you to:
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

22. 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

23. 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

24. Comparison of gating controls

25. 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

26. Outline QC your data This will allow you to:
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

27. 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!

28. 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
IFNg FITC
IL-2 PE

29. 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

30. Outline Configure your instrument Characterize your instrument
Design your panel
Optimize settings for your panel
Run appropriate controls
QC your data

31. 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

32. References
Maecker, H. T., Frey, T., Nomura, L. E., and Trotter, J Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62: 169.
Maecker, H. T., and Trotter, J Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69: 1037.
Roederer, M How many events is enough? Are you positive? Cytometry A 73:
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 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:

33. Acknowledgements Laurel Nomura Margaret Inokuma Maria Suni
Maria Jaimes
Smita Ghanekar
Jack Dunne
Skip Maino
Joe Trotter
Dennis Sasaki
Marina Gever