Resolving 31 colors on a standard 3-laser full spectrum flow cytometer for immune monitoring of human blood samples

Immune monitoring of patients on a single-cell level is becoming increasingly important in various diseases. Due to the often very limited availability of human specimens and our increased understanding of the immune systems there is an increasing demand to analyze as many markers as possible simultaneously in one panel. Full spectrum flow cytometry is emerging as a powerful tool for immune monitoring since 5-laser instruments enable characterization of 40 parameters or more in a single sample. Nevertheless, even if only machines with fewer lasers are available, development of novel fluorophore families enables increasing panel sizes. Here, we demonstrate that careful panel design enables the use of 31-color panels on a 3-laser Cytek® Aurora cytometer for analyzing human peripheral blood leukocytes, without the need for custom configuration and using only commercially available fluorochromes. The panel presented here should serve as an example of a 31-fluorochrome combination that can be resolved on a 3-laser full spectrum cytometer and that can be adapted to comprise other (and possibly more) markers of interest depending on the research focus.