Calphostin c

Purified Treg were resuspended in RPMI 1640 medium supplemented with 10% FBS, 50 uM 2-ME, penicillin/streptomycin, and L-glutamine. Cells were stimulated with plate-bound mouse anti-CD3 and anti-CD28 (1 ug/ml each) in 96-well plates and incubated at 37°C in a cell culture incubator for the indicated times. For Treg proliferation assay, cells were labeled with Cell Trace (Thermo Fisher Scientific) and stimulated with microbeads coated anti-mouse CD3 /CD28 (Dynabeads mouse cell activator). Cell proliferation was determined after 3 days of activation. PKC inhibitor, Calphostin C (PKF) was purchased from Cayman Chemical Company. Tregs were cultured in the presence of various concentrations of PKF in 96-well plates coated with anti-CD3 and anti-CD28 (1 ug/ml each) for 3 days. Supernatant was collected for ELISA. For intracellular cytokine staining, cells were harvested and re-stimulated with PMA (15 ng), ionomycin (1 uM), and Golgistop for 5 hr. Cells were harvested, washed with 1X PBS, and then stained for live cells with Live-dead aqua stain followed by staining for flow cytometry.

Calphostin C was obtained from Cayman Chemicals (Ann Arbor, MI, USA). Phorbol 12-myristate 13-acetate (PMA) was purchased from InvivoGen (San Diego, CA, USA). Diphenyleneiodonium chloride (DPI) and Imipramine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rottlerin and ET 18-OCH3 were purchased from Tocris (Minneapolis, MN, USA). Dextran was obtained from Thermo (Waltham, MA, USA) and VWR (Radnor, PA, USA). Antibodies were purchased as previously described [9 (link),20 (link)].

All reagents used in the experiments were purchased from Cayman Chemicals (Ann Arbor, MI, USA): leukotriene D4 (LTD4) (Cat# 20310), REV 5901 (Cat# 70600), calphostin-C (Cat# 15383), REV 5901 (Cat# 70600), phorbol 12-myristate 13-acetate (Cat# 10008014), BAPTA-AM (Cat# 15551), Amlodipine (Cat#14838). All reagents were dissolved in DMSO except LTD4, which was dissolved in ethanol to make the stock solutions. The final working concentration of the solutions contained less than 0.5% (v/v) of DMSO or ethanol. The toxicity of all the drugs was assessed, and the safest dose was used for further experiments (Figure S1).