2.5. LDH assay

The toxic effect of glutamate on PC12 cells was also determined by lactate dehydrogenase (LDH) release assay (Thermo Scientific) as per the manufacturer’s instructions. PC12 cells were treated with L-glutamate and Bh and AA at different concentrations for 24 and 48 hrs. After treatment, cells were spun down by centrifugation at 2500 g for 5 min at 4 °C. Supernatant of cells (50μl) was transferred to a new plate and 50ul of reaction mixture was added and incubated for 30 min at room temperature. 50ul of stop solution was then added and absorbance was measured at 490 nm and 492 nm wavelengths. Toxicity was calculated by the following formula:

Experimental LDH Release (OD490)

Percent cytotoxicity = ---------------------------------------------- X 100

Maximum LDH Release (OD490)