Ldh cytotoxicity detection kit plus test

Cellular membrane integrity was monitored by the permeability assay based on the release of lactate dehydrogenase (LDH) into the media. LDH release from non-tumourigenic cells HEK293, NIH 3T3 and Human colon adenocarcinoma LS174 cells was determined by using the LDH Cytotoxicity Detection Kit-PLUS test (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, cells were seeded in 12-well plates (10⁵cells/well) and cultured for 72 h with different concentrations (5, 10 and 20 µg/ml) of quince polyphenolic extracts (Peph and Puph). The percentage release of LDH was calculated from the treated cells by comparing it with the maximum release obtained by 1 % Triton X-100 treatment (positive control) and the spontaneous LDH release (mock-treated cells considered as a negative control) as follows: Cytotoxicity (%) = (experimental value–negative control)/(positive control–negative control) × 100.

MTT assay
U87 cells were seeded at 10⁴ or 5 × 10³ cells per well overnight and then treated by different concentrations of the synthetic molecules for 24 or 72 h. Then, cells were incubated for 3 h with MTT (0.5 mg/mL). Afterward, DMSO was added to solubilize formazan crystals and the OD was measured at 600 nm to quantify the percentage of living cells. IC₅₀ were calculated using Prism Graphpad software.
LDH release assay
Cellular membrane integrity was monitored by the permeability assay based on the release of lactate dehydrogenase (LDH) into the media. LDH release from U87 cells was determined by using the LDH Cytotoxicity Detection Kit-PLUS test (Roche Applied Science, Mannheim, Germany) according to the manufacturer protocol. Briefly, cells were seeded at 3 × 10⁵ in 6-wells plate and treated by the synthesized molecules, at their respective IC_50, or Triton X-100, as positive control, for 24 h. The concentration of LDH was evaluated by the measuring the absorbance at 460 nm, after addition of a mixture of substrate and dye solution provided by the kit.