Advanced rpmi 1640 media

Chemosensitive (n = 5) and chemoresistant (n = 7) primary cells were obtained from ascites of high grade OC patients before and after chemotherapy treatment as previously described [103 (link)]. Chemoresistance was determined when patients relapse and no longer respond to chemotherapy. Chemosensitivity was classified as patients responding to chemotherapy and not progressing within 6 months of completing their treatment. These primary cells were grown in Advanced RPMI 1640 media (Gibco, Waltham, MA, USA, cat no. 12633-020) containing 10% fetal calf serum (FCS) (Scientifix, Clayton, VIC, Australia) and 1% each of penicillin/streptomycin (Life Technologies, Mulgrave, VIC, Australia), fungizone (Sigma−Aldrich, St. Louis, MO, USA) and glutamax (Life Technologies, Mulgrave, VIC, Australia) and maintained at 37 °C in a 6% CO₂ environment.
Human HGSOC cell lines, OVCAR-3 and OV-90, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultured in RPMI 1640 (Gibco, Waltham, MA, USA, cat no. 11875-093) with FCS (5% for OVCAR-3, 10% for OV-90), 1% penicillin/streptomycin and 1% glutamax.

Human embryonic kidney 293T cells (HEK293T; ATCC #CRL2316) were cultured in Dulbecco's modified Eagle's medium (DMEM) media (Gibco) supplemented with 10% fetal bovine serum (Gibco). Rat adrenal phaeochromocytoma PC12 cells (PC12; ATCC #CRL-1721) were maintained in advanced RPMI-1640 media (Gibco) supplemented with 10% horse serum, 5% fetal bovine serum, and 1% L-glutamine (all from Gibco). Cells were plated on poly-D-lysine–coated 24-well plates or coverslips and incubated at 37 °C/5% CO₂.
HEK293T and PC12 cells were transfected using PEI (24 (link)) and Lipofectamine 2000 reagent (Invitrogen Ltd), respectively, using a ratio of 2 μl PEI/Lipofectamine per μg DNA. For click chemistry assays, HEK293T cells were transfected with 0.33 μg of pEGFP-Spry2 and 0.66 μg of HA-zDHHC (or pEF-BOS-HA as a negative control) constructs per well of a 24-well plate. For immunoprecipitation assays, HEK293T cells were cotransfected with 0.4 μg of pEGFP-substrate constructs and 0.6 μg of HA-zDHHC constructs per well of a 24-well plate. HEK293T cells were used approximately 20 h post-transfection.
For confocal imaging experiments, PC12 cells were transfected with 0.2 μg of each plasmid per well of a 24-well plate that contained a poly-D-lysine–coated coverslip. PC12 cells were used approximately 44 h post-transfection.