Phorbol 12 myristate 13 acetate

Adenosine 5’-diphosphate (ADP), thrombin-6 receptor activating peptide (TRAP-6), and collagen were purchased from Sigma-Aldrich (St. Louis, Missouri/MO, U.S.A). Phorbol 12-myristate 13-acetate (PMA) was obtained from Tocris-Cookson. Antimycin A was obtained from Genetica y Tecnologia spa (GENYTEC, Chile). Standards for caffeic acid, myricetin, rutin, quercetin, kaempferol, kuromanin chloride (cyanidin 3-O-glucoside), and myrtillin (delphinidin 3-O-glucoside) were from Cayman Chemical (Ann Arbor, MI). All used solvents were of HPLC quality and purchased from Burdick and Jackson (Muskegon, MI, USA).

Human promonocytic leukemia U937 cells (ATCC^® CRL-1593.2™, Manassas, Virginia, USA) were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS; Gibco, 10270, Carlsbad, California, USA), and 293T cells from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% FBS at 37°C in a 5% CO₂ incubator. The methods for the differentiation of U937 cells into macrophages as previously described [12 (link)]. Briefly, U937 cells were cultured in RPMI-1640 medium without FBS for 5 hours and then incubated in RPMI-1640 medium, supplemented with 10% FBS, and 100 ng/mL phorbol 12-myristate 13-acetate; (Tocris Biotechne, 1201, Shanghai, China), for 48 hours. When approximately 70% of the cells became adherent and appeared macrophage phenotype, phosphate-buffered saline was used to wash the non-adherent cells twice. The U937-derived macrophages were used to perform further assays.