4.6. Lactate Dehydrogenase (LDH)-Release Assay

The LDH release assay was performed by the CytoTox 96 non-radioactive cytotoxicity assay kit (Promega; Charbonnie’res-les-Bains, France) according to the manufacturer’s protocol as previously described [54]. Briefly, cells seeded in 96-well plates (5 × 10³ cells/well/100 μL) were incubated with various concentrations of Brevinin-1RL1 or equivalent DMSO for 24 h. Then, cells were lysed for 45 min served as the spontaneous and maximum LDH activity controls for 45 min before running the assay. After centrifugation of the microtiter plates, 50 μL of supernatant was transferred into new tube from each well and mixed with equal volume of supplied reaction mixture and incubated at 37 °C for 30 min. A total of 50 μL of supplied stop solution was added to each well and the absorbance of the colored formazan was determined using the microplate reader (Perkin-Elmer Life Sciences, Waltham, MA, USA) at 450 nm wavelength. Cell membrane integrity was evaluated by measuring the LDH activity released and the assay was repeated in triplicate.