Minimum essential medium (mem)

Human cell cultures of tumor origin—HeLa (cervical tumor), HuTu 80 (human duodenal adenocarcinoma), ChangLiver (human liver HeLa-like line), and normal cell line WI38, VA 13 subline 2RA is a diploid human cell strain composed of fibroblasts derived from healthy lung tissue provided by The Gamaleya National Center for Epidemiology and Microbiology, as well as the Institute of Cytology of the Russian Academy of Sciences. They were grown in a nutrient medium DMEM (PanEco, Moscow, Russia) and MEM (PanEco, Moscow, Russia), containing fetal bovine serum (10% by volume) (ThermoFisher Scientific, Paisley, UK), Glutamax (2 mM) (Gibco, Scotland, UK), and penicillin-streptomycin (1% by volume) (PanEco, Moscow, Russia). The cultivation was carried out at 37 °C in a humidified CO₂ atmosphere (5%).

HeLa cells were cultured at 37 °C (5% CO₂) in DMEM (PanEco, Moscow, Russia) supplemented with 10% fetal bovine serum (BioSera, Nuaille, France), 100 U/mL penicillin, and 100 mg/mL streptomycin (PanEco). For the live cell imaging experiments, the DMEM was replaced by imaging media: MEM (PanEco) supplemented with 10% fetal bovine serum (BioSera) and 20 mM HEPES (Corning, New York, NY, USA).

Cell viability was determined by MTT test as described earlier [54 (link)]. Cells were seeded in a 96-well plate in the amount of 1 × 10⁴ cells/200 µL and cultured at 37 °C in a humidified CO₂ atmosphere (5%) in a nutrient medium DMEM (PanEco, Moscow, Russia) and MEM (PanEco, Moscow, Russia). After 24 h of incubation, 2 mL aliquots of test compounds (0.1 to 100 mM) dissolved in DMSO were added to the cell cultures, and cells were cultured under the same conditions for 24 h. The final content of DMSO in the well did not exceed 1% and did not have a toxic effect on the cells. DMSO also was added to the control wells in a volume of 1%.
After 24 h, 20 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL) was added to each well and the plates were additionally incubated for 2 h.
Using a plate analyzer (Cytation3, BioTek Instruments Inc., Winooski, VT, USA), the optical density was determined at 530 nm. The concentration value causing 50% inhibition of cell population growth (IC₅₀) was determined from dose-dependent curves.