Steps to Success with Multicolor Flow Cytometry

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Steps to Success withMulticolor Flow Cytometry

• Holden T. Maecker

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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

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Outline

• 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

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How do we measure performance?
Resolution Sensitivity
D
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

• Green laser more efficient for PE and PE tandems
• Green laser less efficient for FITC, PerCP and GFP

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Second Example Filters and Spillover
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Outline

• 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

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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
• Linearity, CVs, laser alignment
• PMT voltages required to hit target values
• Data can be visualized in Levey-Jennings plots

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Outline

• 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

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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.
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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

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False positives due to tandem degradation
A.
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
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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
150
Spillover
100
50
0
0
1
2
4
6
8
24
48
Hours of light exposure
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Antibody titration basics

• For most purposes, the main objective is to
  maximize signalnoise (pos/neg separation)
• This may occur at less than saturated staining
• This may or may not be the manufacturers
  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.)

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Antibody titration example
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Outline

• 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

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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

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Experiment-specific setup for existing panel

• Set voltages to achieve experiment-specific
  target channels
• Run single-stained CompBeads and calculate
  compensation
• Run samples

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Outline

• 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

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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

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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

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Comparison of gating controls
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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

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Outline

• 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

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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!

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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
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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

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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

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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

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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.

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Acknowledgements

• Laurel Nomura
• Margaret Inokuma
• Maria Suni
• Maria Jaimes
• Smita Ghanekar
• Jack Dunne
• Skip Maino

• Joe Trotter
• Dennis Sasaki
• Marina Gever