LDH Cytotoxicity Assay

Due to the different aspects measured by cell viability assays, authors have chosen LDH release and neutral red uptake assays. The cytotoxicity detection kit is a colorimetric assay for the quantification of cell death and cell lysis based on the release of lactate dehydrogenase (LDH) from the cytosol of damaged cells into the surrounding medium [36]. An increase in the amount of dead or plasma membrane-damaged cells results in an increase in LDH activity in the culture medium. The cells were plated on 96-well plates for the assays. After 6 and 24 h of treatment with 10 μM TCS, 10 μM MK-801 (NMDA receptor antagonist) or 1 mM L-glutamate (NMDA receptor agonist), 100 μL of the culture supernatants was collected to estimate the LDH and plates with cells were frozen at − 80 °C to measure caspase-3 activity. To measure cytotoxicity, the reaction was stopped after 30 min by adding 1 N HCl and absorbance at a wavelength of 490 nm was measured using the ELISA microplate reader manufactured by Bio-Tek Instruments (Biokom).