Canto 2 flow cytometer instrument

The glioma cells (U87MG) were cultured at a seeding density of 1 × 10⁴/cm² in 6 well plates (TPP. Sigma-Aldrich) for 24HR. Next, the culture media were exchanged with treatments (100%SFCM and 100%FBSCM). The experiment was conducted for 96HR with the exchange of fresh treatment every 48HR and cell cycle analysis was performed at 96HR time point. Cells growing in standard culture media and serum-free media were kept as controls. Cell cycle analysis for glioma cells treated with two types of CM was performed with PI staining according to the manufacturer’s instructions (Lifesciences). Canto BD II flow cytometer instrument (BD, United States) was used for running the samples. The experiment was conducted from six biological samples twice (n = 2) and the results are provided as averages of two independent experiments.

Surface marker characterization for MSCs isolated from all patients was performed in accordance with the International Society for Cellular Therapy (ISCT) recommendations [12 (link)]. BD Stemflow™ hMSC Analysis Kit (BD, USA) was used according to the manufacturer instructions. Passage 3 cells were stained with antibodies against CD73, CD90, CD105, CD44, CD34, CD11b, CD19, CD45, and HLA-DR. Corresponding mouse isotype antibodies were used as a control. Canto BD II flow cytometer instrument (BD, USA) was used for running samples. Diva software (BD, USA) was used for data analyses. The percentage of expressed cell surface markers was calculated from 10,000 gated cells.