Cell Proliferation Assay (MTT) of Glioma Cells and Fibroblasts

The proliferation rates of the glioma cell line (U87MG) and human foreskin fibroblast cell line (DF1) were evaluated by MTT assay. In brief, the glioma cells were cultured at a seeding density of 5 × 10³/cm² in 100 µL of culture media in 96 well plates (TPP, Sigma- Aldrich, United States). After 24HR the media were exchanged with treatments (Cell lysate, 100% CM, and 50% CM). Standard culture media and serum-free media were kept as controls. Media were exchanged every 48HR with respective treatment. The proliferation rate was determined at 24HR, 48HR, 72HR, 96HR and, 120HR by the reduction of (3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) solution according to the manufacturer's instruction. Optical density was measured at 560 nm using a microplate reader (GloMax®-multi, Promega, United States). Each proliferation (MTT) experiment was repeated three times (n = 3). Serum-free media (SFM as DMEM-F12 only) was kept as a control for SFCM and FBS media (DMEM-F12 + 10%FBS) was kept as a control for cell lysate and FBSCM. Glioma cells (U87MG) at passages 3-5 were used for all experiments. Similarly, fibroblasts (P3) were cultured at the same seeding density as glioma cells and after 24HR the media were exchanged with treatments {(Cell lysates and 100%CM only (SFCM, FBSCM)} from two types of MSCs. The rest of the procedure for MTT was being followed as described above for glioma cells.

Note. At this point, we found that cell lysates induced a generalized inhibitory effect on cancer cells and fibroblasts. While conditioned media induced a variable effect on fibroblasts. Therefore, further experiments were performed only with 100% CM since CM exhibited a selected effect on cancer cells.