Umbralisib

Phospho-specific flow cytometry with fluorescence cell barcoding were performed as has previously been reported.^{17 (link)} Briefly, cells (either patient samples or cell lines) were plated at 1×10⁶ cells/mL and incubated with either DMSO, umbralisib (Selleck Chemicals, S8194) (20 μM), ruxolitinib (Selleck Chemicals, S1378) (60 nM), or the combination for 30 min. Cells in the stimulated condition were subsequently stimulated with 20 ng/mL GM-CSF (Peprotech, 300–03-20ug) for 15 min and all were then exposed to 50x Ax700 (Invitrogen, A20010) viability stain. Cells were fixed in 1.6% PFA (Fisher Scientific, 50–980-487) in dark at RT for 10 min, washed with PBS, and permeabilized with MeOH (Fisher Scientific, A4121) and stored in −80 °C. Cells were washed twice with PBS before barcoding with serial dilutions of Pacific Orange (1:2.5) (Invitrogen, P30253) and Pacific Blue (1:2.4) (Invitrogen, P10163) dyes and combining in a single tube.^{18 (link),19 (link)} Cells were stained with pSTAT5-PE/Cy7 (Clone 47, BD:560117), pERK-Ax488 (Clone 6B8B69, Biolegend:369508), pAKT-Ax488 (Clone M89–61, Company BD:560404 and Clone D9E, CST:5315S) and pS6-Ax647 (Clone D57.2.2E, Company CST:4851S) in the dark at RT for 30 min. Cells were washed with 1mL PBS/BSA and resuspended in 0.2 mL PBS/BSA and run a 4-laser LSR Fortessa (Becton Dickinson).