Lactate dehydrogenase activity colorimetric assay kit

Intracellular LDH activity was assessed using the Lactate Dehydrogenase Activity Colorimetric Assay Kit (#K726; BioVision) according to the manufacturer's instructions. In brief, treated MM cell lines were cultured at 2 × 10⁵ cells·mL⁻¹, and then, 1 × 10⁶ cells were harvested for cell lysate preparation. In this colorimetric assay, LDH reduces NAD to NADH and then interacts with a probe to produce a color at a wavelength of 450 nm (λ_max = 450 nm), which was measured using a spectrophotometer. The data were calculated as the LDH activity per cell lysate protein amount and expressed as unit·mg⁻¹ (Fujiwara et al., 2013; Jin et al., 2017).

Cell viability was assessed by the mitochondria-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) to formazan, as described previously^{30 (link)}. Briefly, once supernatants were collected, the remaining cells were incubated for 2 h in 250 μl/well serum-free culture medium containing 1 mg/ml MTT; the medium was then removed and the cells lysed with 250 μl/well dimethyl sulfoxide (Sigma); and, optical density (OD₅₇₀) measured using a POLARstar Omega microplate reader.
The kinetics of cell survival was examined by enzymatic reduction of resazurin to resorufin using the Alamar Blue cell viability assay (Pierce, Rockford, IL, USA)^{34 (link)}. Fluorescence was measured at 545/590 nm (excitation/emission) using a POLARstar Omega microplate reader, with data expressed as the percentage of control cell survival.
Total cellular DNA concentrations were measured using the CyQuant Cell Proliferation Assay (Thermo Fisher Scientific, Loughborough, UK). Fluorescence was measured at 480/530 nm (excitation/emission), with data expressed as arbitrary fluorescence units.
Leakage of cellular LDH was measured in cell culture supernatants using the Lactate Dehydrogenase Activity Colorimetric Assay Kit (Biovision, California, USA), according to the manufacturer’s instructions.