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Design and optimization of multicolor panels

Outline. Choosing fluorochrome combinations and filter setsMatching antibody specificities with fluorochromesControls and standardization. Outline. Choosing fluorochrome combinations and filter setsChoose bright fluorochromesMinimize spillover between detectors. Bright" = good resolution sens

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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. “Bright” = good resolution sensitivity

    5. Various fluorochromes-stain index

    6. Spillover affects resolution sensitivity

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

    11. Spillover affects resolution sensitivity

    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

    14. New tandems will be more stable APC-H7 as a replacement for APC-Cy7:

    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 A 62, 169. Maecker, H. T., and Trotter, J. (2006).Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69, 1037.

    21. Acknowledgements Laurel Nomura Margaret Inokuma Maria Suni Smita Ghanekar Daiva Gladding Jack Dunne Skip Maino

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