Design and optimization of multicolor panels

Holden T. Maecker

Outline

• • • Choosing fluorochrome combinations and filter sets Matching antibody specificities with fluorochromes Controls and standardization

Outline

• Choosing fluorochrome combinations and filter sets • Choose bright fluorochromes • Minimize spillover between detectors

“Bright” = good resolution sensitivity D W2 W1

Stain Index (SI)



D W

Where D = difference between positive and negative peak medians, and W = 2 x rSD (robust standard deviation)

Various fluorochromes-stain index Reagent

PE Alexa 647 APC PE-Cy7 PE-Cy5 PerCP-Cy5.5

PE-Alexa 610 Alexa 488 FITC PerCP APC-Cy7 Alexa 700 Pacific Blue AmCyan

Clone

RPA-T4 RPA-T4 RPA-T4 RPA-T4 RPA-T4 Leu-3a RPA-T4 RPA-T4 RPA-T4 Leu-3a RPA-T4 RPA-T4 RPA-T4 RPA-T4

Filter

585/40 660/20 660/20 780/60 695/40 695/40 610/20 530/30 530/30 695/40 7801/60 720/45 440/40 525/50

Stain Index

356.3

313.1

279.2

278.5

222.1

92.7

80.4

75.4

68.9

64.4

42.2

39.9

22.5

20.2

Spillover affects resolution sensitivity

FITC background contributes noise to PE measurement www.bdbiosciences.com/spectra

Choices for 6-, 8-, 10-, and more colors

6-color FITC or Alexa 488 PE PerCP-Cy5.5

PE-Cy7 APC or Alexa 647 APC-Cy7 8-color FITC or Alexa 488 PE PerCP-Cy5.5

PE-Cy7 APC or Alexa 647 APC-Cy7 AmCyan Pacific Blue 10-color FITC or Alexa 488 PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5

PE-Cy7 APC or Alexa 647 Alexa 680 or 700 APC-Cy7 AmCyan Pacific Blue Additional FITC or Alexa 488 PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5

PE-Cy7 APC or Alexa 647 Alexa 680 or 700 APC-Cy7 AmCyan Pacific Blue Q dot 655, 705…

Outline

• • • Choosing fluorochrome combinations and filter sets Matching antibody specificities with fluorochromes Controls and standardization

Fluorochrome selection considerations

• “Bright” antibodies go on “dim” fluorochromes • Avoid spillover from bright cell populations into detectors requiring high sensitivity • Take special care with tandem dyes

Data spread of fluorescent signals

compensated uncompensated data spread uncompensated compensated FITC mean fluorescence --------------------------- negative positive ---------- 123 123 --------- 3541 3564 PE mean fluorescence --------------------------- negative positive ---------- 184 134 --------- 1648 136 HT Maecker, T Frey, LE Nomura, J Trotter:

Selecting fluorochrome conjugates for maximum sensitivity

.

Cytometry A

2004,

62

:169-73.

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.

Special requirements of tandem dyes

• Compensation requirements for tandem dye conjugates can vary, even between two experiments with the same antibody • Require experiment-specific compensation • Certain tandem dye conjugates (APC-Cy7, PE-Cy7) can degrade with exposure to light, elevated temperature, and fixation • • Minimize exposure to these conditions Use BD Stabilizing Fixative for final fixation

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

B.

Without CD8 APC-Cy7: False positives in APC channel reduced in absence of APC-Cy7 False positives in PE channel remain

New tandems will be more stable

• APC-H7 as a replacement for APC-Cy7: 250 200 150 100 50 0 0 Comparison of Sample Stability (in BD Stabilizing Fixative at RT) 1 2 4 6 Hours of light exposure 8 24 48 CD4 APC-Cy7 CD8 APC-Cy7 CD4 APC-H7 CD8 APC-H7

Outline

• • • Choosing fluorochrome combinations and filter sets Matching antibody specificities with fluorochromes Controls and standardization

Types of controls

• • •

Instrument setup controls

• PMT voltage settings • Compensation (per experiment)

Gating controls

• Isotype controls • Fluorescence-minus-one (FMO) controls

Biological controls

• Unstimulated samples • Healthy donors

Comparison of gating controls

Standardization using lyophilized reagents

• • • • • Lyophilization provides increased stability, even at room temperature or 37 o C 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

Conclusions

• Polychromatic flow cytometry is not impossible • Select fluorochromes for brightness and least spillover • Optimize antibody panels by taking into account reagent brightness and data spread • Stabilize longitudinal experiments with proper QC • Some solutions that can help • • • Lyophilized reagent plates Stabilizing fixative Beads for calibration and compensation

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.

Acknowledgements

• • • • • • • Laurel Nomura Margaret Inokuma Maria Suni Smita Ghanekar Daiva Gladding Jack Dunne Skip Maino • • • Joe Trotter Dennis Sasaki Marina Gever