Phorbol n myristyl acetate

Conditions were derived from prior mass cytometry signaling studies of healthy human bone marrow.^{4 (link)} Briefly, cryopreserved cells were thawed into RPMI containing 10% fetal bovine serum, heparin (20 U/mL, Sigma, St. Louis, MO), and benzonase (25 mU/mL, Sigma), and labeled with cisplatin (2.5 μM, Enzo Life Sciences, Farmingdale, NY) in serum free RPMI.^{34 (link)} cisplatin-treated cells were incubated in serum-containing RPMI for 30 minutes, followed by 1 hour incubation in the presence or absence of inhibitor (5 μM ruxolitinib or 2 μM IKK inhibitor VII), followed by 15 minute stimulation with cytokine and subsequent fixation and staining. Stimulation conditions in the screening experiment (Figure 1) were: basal (unstimulated), ruxolitinib, thrombopoietin (TPO, 50 ng/mL), TPO plus ruxolitinib, granulocyte colony stimulating factor (G-CSF, 20 ng/mL), and sodium pervanadate (125 μM). Subsequent experiments also included 2 μM IKKiVII, TNFα (20 ng/mL), and IKKiVII plus TNFα. Cytokines were obtained from Peprotech (Rocky Hill, NJ), ruxolitinib from Chemie-Tek (Indianapolis, IN), and IKKiVII from EMD Millipore (Billerica, MA). Intracellular TNFα profiling (Figure 6) included secretion inhibitor (brefeldin A/monensin, Ebioscience, San Diego, CA), and stimulation with phorbol-N-myristyl-acetate (PMA, Invivogen, San Diego, CA) and ionomycin (EMD Millipore).