Great ICOS Staining at Exceptional Value

Results Summary

The fluorochrome BV750 may be detected in the “BV785/786-channel” or detected separately from these fluorochromes, depending on detector configuration/ flow cytometer setup. Here, samples were acquired on a 5L Cytek Aurora which was able to separate between BV711, BV750 and BV785 signals without much spillover. ICOS is upregulated upon T cell activation. The signal is sometimes difficult to detect, but we were able to detect a distinct ICOS-positive population of CD4+ T cells even at a titer of 1:400 (shown here). BV750 is a among the brighter BV-fluorochromes, which aided with the good signal. The staining here was performed on murine splenocytes, however, we were also able to successfully stain human cells with this same antibody (clone C398.4A).In the following, you will find my staining protocol in short. Incubation steps were performed at 4°C in the dark. 1.Count and adjust splenocytes to 1.5E6 cells, plate in a 96-well plate. 2.If required, stain cells with viability dye. Wash. 3. Stain with extracellular master mix for 20 min. Wash 2x. 4. Fixate/ Permeabilize with buffer of choice, depending on application. I used ThermoFisher FoxP3 fixation/permeabilization kit for 20 min. Wash with perm/ Wash 2x. 5. Perform intracellular staining for 20 min. Wash 2x.