Combinatorial antibody titrations for high‐parameter flow cytometry

Abstract The objective of titrating fluorochrome‐labeled antibodies is to identify the optimal concentration for a given marker‐fluorochrome pair that results in the best possible separation between the positive and negative cell populations, while minimizing the background within the negative population. Best practices in flow cytometry dictate that each new lot of antibody should be titrated on the sample of interest. However, many researchers routinely use large (30+) color panels due to recent technical advancements in fluorescence‐based cytometry instrumentation which quickly leads to an unmanageable number of individual titrations. In this technical note, we provide evidence that antibodies can be effectively titrated in groups rather than individually, resulting in considerable time and cost savings. This approach streamlines the process, without compromising data quality, thereby enhancing the efficiency of setting up high‐parameter cytometry experiments.