Gmem media

African green monkey kidney (Vero) cells were grown in OptiMEM (Gibco-Life Technologies) supplemented with 5% fetal bovine serum (FBS, Hyclone) and 1% L-glutamine (Gibco-Life Technologies) at 37°C with 5% CO₂. Baby hamster kidney cells constitutively expressing the T7 polymerase (BSR T7/5) [56 (link)] were grown in GMEM media (Gibco-Life Technologies) supplemented with 10% FBS and 2% MEM amino acids (Gibco-Life Technologies). Every second cell passage, 2% of gentamicin (Quality Biological) was added to maintain selection for the T7-polymerase-expressing cells.

African green monkey kidney (Vero) cells were grown in OptiMEM (Gibco-Life Technologies) with 5% fetal bovine serum (FBS, Hyclone) and 1% L-glutamine (Gibco-Life Technologies). Vero cells were maintained in 2% FBS during experiments. Baby hamster kidney cells that constitutively express the T7 polymerase (BSR T7/5) [43 (link)] were grown in GMEM media (Gibco-Life Technologies) with 10% FBS and 2% non-essential amino acids (Gibco-Life Technologies). Every other cell passage, 2% of gentamicin (Quality Biological) was added to the media to maintain selection for the T7-polymerase-expressing cells. Both cell lines were maintained at 37°C with 5% CO₂.

Total glutathione was measured using an enzymatic recycling method as described (Irfan Rahman AKSKB, 2006 ). Briefly, HepG2 and HEK293A cells were cultured for 48h in GMEM media (Gibco) supplemented with 10% FBS, 0.4 M serine and 0.4 M glycine, 2 mM glutamine, penicillin, and streptomycin. Cells were washed extensively with ice‐cold PBS, lysed by freeze‐thaw cycles, and the lysates cleared by centrifugation. After the conversion of the glutathione in the lysate to the reduced form (GSH), total GSH content was measured using 5,5'‐dithiobis‐(2‐nitrobenzoic acid) DTNB. DTNB is reduced to 2‐nitro‐5‐thiobenzoate which was quantified by absorbance at 412 nm in CLARIOstar (BMG LabTech) 96‐well plate reader. Total GSH was normalized to the amount of protein in a separate set of samples plated, processed, and harvested at the same time.
In some experiments, total glutathione was measured using the GSH/GSSG‐Glo kit (Promega). Cells were plated at 10000 cells/well in a 96‐well white wall, clear‐bottom plate, and cultured for 48h in GMEM media supplemented with 10% FBS, 0.4 M serine, 0.4 M glycine, 2 mM glutamine, penicillin, and streptomycin. After washing in ice‐cold, calcium/magnesium‐free PBS, total glutathione levels were measured using the instructions provided by the manufacturer. The results presented for glutathione measurements are representative of three independent experiments.