2.8.2. Lactate dehydrogenase (LDH) cell cytotoxicity assay

The evaluation of cytotoxicity by LDH assay was performed by obeying previously described method with slight modification (Zhang and Wang, 2013). Briefly, 6 well plates was seeded with 2 × 10⁵ HeLa cells per well and incubated at 37 °C for 24 h in a CO₂ humidified incubator until the cells attained confluent growth. After 24 h incubation, the cells were treated with different doses (50, 250, 500, and 1000 µgmL⁻¹) of test samples and positive control (quercetin), followed by 24 h incubation at 37 °C. After the exposure, 100 µL of supernatant from each well was taken and mixed with 2 mL of Tris-EDTA-NADH buffer and incubated for another 30 min at 37 °C. After this, freshly prepared sodium pyruvate (400 µL) was added to the reaction mixture and cell viability was measured at 340 nm after every 15 sec for 3 min.