Glutamate-induced excitotoxicity and MTT cell viability assay

SH-SY5Y neuroblastoma cells, a cell line derived from the SK-N-SH neuroblastoma cells (Key Gen Biotech Co. Ltd. Jiangsu, China), were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, 04–001-1ACS, Israel) at a constant temperature at 37 °C with 5% CO2. The cells were plated in 96-well plates (density: 5 × 103/well) until cell confluence reached ~ 60%. Cells were then challenged with different concentrations of glutamate (1, 2, 4, 8, 10, 20, 40, 80 mM) and 100 μM D-serine (a NMDAR co-agonist) for 24 and 48 h; ST2-104 (0.1, 1, 5, 10, 25, 50, 100 μM) for 24 h and 48 h; or Glu (20 mM) and 100 μM D-serine with ST2-104 peptide (3, 10, 30 μΜ; pre-treatment 30 min) for 24 h.

Following these treatment conditions, the culture medium was discarded and 20 µl 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, USA) solution was added to each well at 37 °C for 4 h. DMSO (150 μl/well) was added for 10 min to dissolve the purple crystals; MTT is a yellow tetrazolium dye that turns purple when it is reduced to an insoluble formazan with DMSO. Finally, the optical density values were examined using a microplate reader (Tecan, Switzerland) at 490 nm.