Rpmi 1640 cell culture medium

HeLa cervical cancer cells were purchased from Highveld Biological, Johannesburg, South Africa. RPMI 1640 cell culture medium and fetal bovine serum was purchased from GE Healthcare Life Sciences (Logan, UT, USA). Trypsin-EDTA, Dulbecco’s phosphate buffered saline (DPBS) with Ca²⁺ and Mg²⁺ and Dulbecco’s phosphate buffered saline (DPBS) without Ca²⁺ and Mg²⁺ were purchased from Lonza (Wakersville, MA, USA). Trypan blue, bisBenzamide H 33,342 trihydrochloride (Hoechst 33342), cisplatin, penicillin/streptomycin and bovine serum albumin fraction V were purchased from Sigma-Aldrich (St. Louis, MI, USA). NucRed^TM Live 647, CellRox^® Orange reagent, Lysotracker™ Deep Red and Tetramethylrhodamine ethyl ester (TMRE) were purchased from Molecular Probes^®-Life Technologies-Thermo Fisher Scientific (Logan, UT, USA). Annexin V-FITC/PI kit was purchased from MACS Miltenyi Biotec (Cologne, Germany). Cleaved caspase 3 (Asp175) (D3E9) Rabbit mAb, Cleaved caspase 8 (Asp391) (18C8) Rabbit mAb, Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa fluor^® 647 conjugate), Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa fluor^® 488 conjugate) and Phosphorylated Histone H3 were purchased from Cell Signaling Technology (Danvers, MA, USA).

The human glioma U251 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). CCM (C₂₁H₂₀O₆; >98% purity) was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) to create a stock solution, and stored at −20°C until use. The human malignant GBM U251 cell line was maintained in RPMI-1640 cell culture medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (all Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) in a humidified incubator containing 5% CO₂ at 37°C. Cultures were passaged every 2–3 days after reaching 80% confluence. Cells in the logarithmic growth phase were used in the experiment.
The cells were divided into the control group (complete culture), the DMSO group (DMSO only, as the negative control group) and the CCM group (treated with CCM at various concentrations, as the treatment group).