Ldh cytotoxicity assay

To check for LDH enzyme activity, the LDH cytotoxicity assay (Promega, Mannheim, Germany) was performed. The assay is based on an enzymatic coupling reaction: LDH oxidizes lactate in order to generate NADH, which then reacts with pyruvate and a dye to generate a yellow color. LDH activity was quantified with a plate reader (VarioSkan Flash Multimode Reader, Thermo Scientific, USA) at 490-nm absorption. In summary, cells were seeded 24 h prior to treatment with 2.5 × 10³ cells/well in 200 µL of serum-free media/well, and incubated with either the indicated concentrations of metformin, diclofenac, or a combination of both; NaOxamat (25 mM) was used as a positive control. Then, 24 h and 48 h later, LDH activity was measured.

PBMC viability, following treatment with RBBV and venom fractions, was determined using a LDH cytotoxicity assay (Promega, Madison, WI, USA). Experiments were performed according to the manufacturer’s instructions. In brief, PBMCs were treated with test compounds or vehicle controls, and the cells cultured at 37 °C and 5% CO₂ for 24 h. Maximum cell lysis control samples were lysed using 10% Triton X-100 lysis buffer 45 min before cell supernatant collection. Following incubation, culture plates were centrifuged at 500× g for 5 min. Culture supernatant was collected, and LDH standards (Sigma-Aldrich) were prepared in duplicate. Samples (25 µL) were mixed with the substrate (25 µL) and incubated for 15 min in the dark at room temperature. To terminate the reaction, we added stop solution (25 µL) to all samples. The plate absorbance was measured at 490 nm using a Gen5 microplate reader (BioTek, Winooski, VT, USA). The LDH sample values, within the linear range of the assay, were converted to ng/mL using the standard curve.