Mtt reagent

The determine the effect of glioma-derived conditioned media (CM) on PC12 cell viability, two million TS603 cells were plated in T75 flask and cultured in proliferation conditions for 3 days. The same culture media were incubated in T75 flask without cultivating cells and used as a control. Samples of culture media were filtered through 0.2 µm filter and kept at -20 until use. PC12 cells were cultured in 96-well plates until cells were approximately 70% confluent. The glioma-derived CM or control media were added to the experimental well in the 96-well plate. 72 h later, the cells were then added with 10 µl of MTT reagent (Trevigen) for 2 h, and solubilized with 100 µl of supplied detergent for 4 h at 37 °C. The cell viability was determined by measuring the absorbance at a wavelength of 570 nm, and the values were normalized using control media-treated cells.

We used the MTT reagent (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s instructions^{10 (link)} to examine cell viability. 2000 cells/well were seeded a 96-well plate. At 24 hours postseeding, the cells viability was measured by MTT for 24 hrs, 48 hrs or 72 hrs, respectively. We further measured the optical density at 570 nm with a microplate reader (Spectral Max250; Molecular Devices, CA, USA).