Reversible, but none of the irreversible, BTK inhibitors affect viability or blocks CD25 expression in isolated B cells from C481S. (A) Viability of B cells as determined by flow cytometry following indicated stimulation and BTK-inhibitor treatment. Cell viability was assessed using annexin V and LIVE/DEAD (viability marker) staining. (B) CD25 expression on B cells as determined by flow cytometry following indicated stimulation and BTK inhibitor treatment. (C) Representative data from 1 of 2 independent experiments. Prior gating is indicated below FACS plots. (A-B) Bars indicate mean with SD. Data are from 2 independent experiments and a total 4 wild-type, 4 C481S, and 2 BTKKO mice were analyzed. (A) Values were normalized to the percentage of live (LIVE/DEAD⁻ Annexin V⁻) cells in the average of wild-type stimulated controls from each independent experiment. Data from BTK KO mice are only shown in panel A. The highest dose of acalabrutinib reduced survival and CD25 expression in C481S mice.