With well-designed panels and high quality reagents, multicolor flow cytometry is a powerful tool for detecting and monitoring multiple parameters of your sample simultaneously. However, the quality of the data is highly dependent upon an optimized panel design and properly tuned instrumentation. These 7 tips can help you improve the quality of your flow cytometry data and avoid common multicolor flow cytometry pitfalls.
Fluorochrome Selection: Not all dyes are created equal! Some dyes are extremely bright, while others are vanishingly dim in comparison. However, choosing the brightest dye is not always the brightest idea, and avoiding a dimmer dye is not always prudent. In general, it is advisable to use a dye that is sufficiently bright to detect the antigen of interest while causing the least spillover or bleed into other detectors. Spillover occurs when the emission spectrum of one fluorochrome is detected by a detector set to measure the emission of a different fluorochrome, thereby contributing to unwanted signal in the channel of the second fluorochrome. Brighter emission means more potential for spillover and more data spreading, which can lead to a loss of resolution. To avoid this, use brighter dyes for weakly expressed antigens and, conversely, use dimmer dyes for strongly expressed antigens. Following this rule will allow you to democratize your dyes!
No/marginal distortion from other dyes
Causing no/marginal distortion in other channels
Loss of sensitivity - Dim positive events
cannot be detected, but are not of interest
Limit of interest
Marked loss of sensitivity -
Most of the dim positive events of the
orange population cannot be detected
Minor loss of sensitivity -
Very dim positive events of the blue
population cannot be detected
Keep these 7 tips in mind as you plan your next multicolor flow cytometry assay, and your panel will reward you with optimum signal detection and increased certainty of results with reduced complexity. To further improve your odds of success with multicolor flow cytometry, use high quality and robust reagents that have undergone the scrutiny of regulatory agencies and manufacturing standards. The more reliable your reagents, the more reliable your data!