Ldh cytotoxicity kit

We incubated the cells in the culture plate with 96 wells at the concentration of ten thousand cells per well. After being incubated for 24 h, cells were incubated with various cuminaldehyde’s concentrations for 48 h. Lactate dehydrogenase’s activity was evaluated by LDH-Cytotoxicity Kit (BioVision, Milpitas, CA, USA) according to the supplier’s instructions. The samples’ absorbance at 490 nm wavelength was evaluated by a spectrophotometer (Tecan infinite M200, Tecan, Männedorf, Switzerland). Data are presented as the percent of activity’s variation relative to untreated control.

Cytotoxicity of bLF and the LF peptides toward HeLa cells was analyzed by monitoring mitochondrial activity. For this, approximately 5 × 10⁴ HeLa cells were seeded in a 96-well plate and cultured serum-free for 16 h. Cells were washed twice with PBS. A serial dilution of bLF and each peptide was made in PBS (0–25 µM). Cells we were incubated at 37 °C under 5% CO₂ for 1 h. Maximal cytotoxicity was induced by adding 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). Cells were washed twice with PBS and incubated in PBS containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, ThermoFisher Scientific, Bleiswijk, the Netherlands) for 2 h and washed again. The MTT-crystals that were precipitated in the cells were resuspended in 100% dimethylsulfoxide (DMSO, Sigma-Aldrich). Absorption was measured at 570 nm with 630 nm for background correction using a Multiscan FC microplate photometer (ThermoFisher Scientific,). In parallel, cytotoxic effects of bLF and the peptides on Hela cells were analyzed by measuring the lactate dehydrogenase (LDH) release using an LDH-cytotoxicity kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions (Sijbrandij et al. 2017 (link)). The experiments were performed in duplicate and repeated three times.