Hyclone mem ebss

The human lung fibroblast WI38 cell line, osteosarcoma cell line SAOS-2 (p53 null and truncated RB1), and the pancreatic cancer cell lines, BxPC-3, AsPC-1, MIA PaCa-2, and PANC-1 were purchased from the American Type Culture Collection (Manassas, VA, USA). WI38 cells were grown in Hyclone MEM/EBSS (ThermoFisher Scientific, Waltham, MA) media supplemented with 10% research grade fetal bovine serum (FBS) (PAA Laboratories, Dartmouth, MA) and 1% Penicillin Streptomycin (Corning, Corning, NY) and SAOS-2, MIA PaCa-2, and PANC-1 cells were grown in Hyclone High Glucose DMEM (ThermoFisher Scientific, Waltham, MA) supplemented with 10% FBS and 1% Penicillin Streptomycin. BxPC-3 and AsPC-1 were cultured in RPMI supplemented with 10% or 15% FBS (respectively) and 1% Penicillin Streptomycin. Cells were cultured at 37°C in a humidified 5% CO₂ incubator.
Ad.CMV (adenovirus with CMV promoter) and Ad.CMV.p53 (Adenovirus containing wild-type p53 gene under control of CMV promoter) viral vectors were generated using the AdEasy system (Carlsbad, CA). The Ad.CMV.pRb (Adenovirus containing RB1 gene cDNA under control of CMV promoter) vector was provided by Dr. Juan Fueyo (M.D. Anderson Cancer Center, The University of Texas). The Ad.GFP and Ad.GFP.RGS16 viruses were purchased from Vector Biolabs (Philadelphia, PA). Viruses were amplified and titered as previously described [81 (link)-83 (link)].

U87MG-Luc2 (U87), primary E2 and G7 glioma cell lines were used in this study. E2 and G7 cells were derived from freshly resected human GBM specimens as previously described [49 (link)]. U87 cells were cultured in Hyclone MEM/EBSS (Fischer Scientific), 10% fetal calf serum, 1% L-Glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen) and 1% sodium pyruvate (Invitrogen). E2 and G7 cells were cultured in MEMα (Gibco) supplemented with 5% L-Glutamine. The U87 cell identity was confirmed using the short tandem repeat (STR) analysis (Identicell, Denmark).
For western blotting and flow cytometry both cell lines were cultured in stem cell enriching conditions in Advanced DMEM F12 medium (Gibco) supplemented with 1% B27 (Invitrogen), 0.5% N2 (Invitrogen), 4 μg/ml heparin, 20 ng/ml fibroblast growth factor (bFGF, Sigma), 20 ng/ml epidermal growth factor (EGF, Sigma) and 1% L-Glutamine. Stem cell enriched cultures were grown on Matrigel (BD Biosciences) coated flasks (1:50 dilution) in serum free media.