RAW 264.7 Macrophage Cell Culture and Reagents

RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC TIB-71, Manassas, VA; Lots 70000171, 70012232 with cell identity confirmed by ATCC via growth properties, morphology, and COI assay). Cell media (supplemented DMEM) was composed of DMEM obtained from Thermo Fischer Scientific (Waltham, MA), fetal bovine serum (FBS, contains ~400 μM BSA as specified by manufacturer) from Millipore-Sigma (St. Louis, MO), penicillin, streptomycin and GlutaminePlus (L-alanyl-L-glutamine) from Atlanta Biologicals (Flowery Branch, GA). Dulbelcco's phosphate buffered saline (D-PBS) was from Gibco (Gaithersburg, MD). All cell media was regularly subjected to mycoplasma testing to ensure lack of infection. Matriplate 96 well imaging plates were from Matrical, Inc. (Spokane, WA). Acetylsalicylic acid (Lot SLBV2290), ibuprofen (Lot SLBX6584), acetaminophen (Lot SLBR2060V), diclofenac (Lot BCBS5961V), wortmannin (Lot 086M4026V), PDGF (Lot SLB63548V), and HEPES (free acid) were obtained from Millipore-Sigma (St. Louis, MO). Gö6976 (Lot 6A/174381) was purchased from Tocris Biosciences (Minneapolis, MN). CellMask Deep Red and Green membrane stains were purchased from ThermoFischer Scientific (Waltham, MA). Cell-culture grade DMSO was purchased from MP Biomedicals (Santa Ana, CA). AKT-PH-mRFP mammalian expression plasmid was subcloned by John H. Evans [26 (link), 30 (link)].