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Design and optimization of multicolor panels

This comprehensive guide covers the design and optimization of multicolor panels for flow cytometry, including selecting fluorochrome combinations and filter sets, matching antibody specificities, controls, and standardization techniques to achieve optimal results. It discusses the importance of choosing bright fluorochromes, minimizing spillover between detectors, and considerations for fluorochrome selection. The guide emphasizes maximizing sensitivity, resolution, and stability for polychromatic flow cytometry experiments through careful panel design and experimental controls.

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Design and optimization of multicolor panels

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  1. Design and optimization of multicolor panels Holden T. Maecker

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

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

  4. W1 W2 Where D = difference between positive and negative peak medians, and W = 2 x rSD (robust standard deviation) “Bright” = good resolution sensitivity D

  5. Various fluorochromes-stain index

  6. Spillover affects resolution sensitivity FITC background contributes noise to PE measurement www.bdbiosciences.com/spectra

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

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

  9. 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

  10. Data spread of fluorescent signals compensated uncompensated data spread FITC mean fluorescence PE mean fluorescence ---------------------------- ---------------------------- negative positive negative positive ----------- ---------- ----------- ---------- uncompensated 123 3541 184 1648 compensated 123 3564 134136 HT Maecker, T Frey, LE Nomura, J Trotter: Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 2004, 62:169-73.

  11. 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.

  12. 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

  13. 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:

  14. CD4 APC-Cy7 CD8 APC-Cy7 CD4 APC-H7 CD8 APC-H7 New tandems will 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

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

  16. 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

  17. Comparison of gating controls

  18. Standardization 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

  19. 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

  20. References • Maecker, H. T., Frey, T., Nomura, L. E., and Trotter, J. (2004).Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A62, 169. • Maecker, H. T., and Trotter, J. (2006).Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A69, 1037.

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

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