4.2. Assay of the Viability and Proliferation of C6 Glioma Cells

Dulbecco’s modified Eagle’s medium (DMEM, Merck KGaA, Darmstadt, Germany) with the addition of antibiotics (penicillin, streptomycin and amphotericin B; all from Merck KGaA, Darmstadt, Germany) and 10% HyClone™ fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) was used for the preparation of cell suspensions with a concentration of 0.20 million cells/mL (for analysis after 24 h), 0.10 million cells/mL (for analysis after 48 h) and 0.04 million cells/mL (for analysis after 72 h). During the experiments, into the wells of one of the 96-well culture plates (Costar, Corning Inc, Corning, NY, USA), 200 µL of a cell suspension with a concentration of 0.20 million cells/mL were seeded; into the wells of the second plate, a concentration of 0.10 million cells/mL; and into the wells of the third plate, a concentration of 0.040 million cells/mL. Afterward, COX or LOX inhibitors, nicotine, nAChR blockers or solvents for these drugs were added at a volume of 25 μL each. Saline was used to dissolve nAChR blockers, DMEM with 10% FBS for nicotine, and DMSO for COX and LOX inhibitors. The required volume was adjusted with DMEM containing 10% FBS. Final concentrations of nAChR blockers were 1 and 100 nM. The following final concentrations COX or LOX inhibitors were used: indomethacin—10 μM, SC-560—0.1 μM, NS-398—5 μM, NDGA—30 μM, zileuton and baicalein—50 μM. A similar amount of solvent was added in the control series. In all wells, the final concentration of DMSO was 0.1%. Nicotine was used at final concentrations of 0.001–10 µL/mL. The combined data of several experiments are shown; in each series, n = 4–6. The plates with cells were kept in a CO₂ incubator at 5% CO₂ and 37 °C. The cells from the first, second and third plates were collected in flow cytometry tubes, after 24, 48 and 72 h, respectively. Staining with propidium iodide (Sigma) was applied to evaluate cell viability. Flow cytometer BD FACSCanto II with Diva 7.0 software (Becton Dickinson, Franklin Lakes, NJ, USA) was used for the cell sample analyses. Cell proliferative activity was determined by calculating the cell concentration in the samples using FLOW-COUNT™ reference fluorospheres (Beckman Coulter, Inc, Brea, CA, USA).