Cytotox onetm homogeneous membrane integrity assay kit

Rupture of the cell membrane and removal of intracellular components to the surrounding medium is a hallmark feature of necrosis. The release of LDH in the medium after SeNP treatment was determined using CytoTox-ONE^TM Homogeneous Membrane Integrity Assay Kit (Promega). LNCaP cells were seeded in a white colored 96-well opaque walled Nunclon Delta surface cell culture plate at a density of 1 × 10³ cells per well in RPMI 3160 medium supplemented with 10 mM HEPES buffer, 10% FBS, and antibiotics. After 24 h of resting period at 37°C in a 5% CO₂ incubator, 2 μg Se/ml SeNPs were added to the test wells and the plate was further incubated for 12, 18, 24, or 30 h at 37°C. CytoTox-ONE^TM assay kit was used according to the manufacturer's protocol. The fluorescent signals corresponding to the LDH release was estimated on a BioTek Power Wave Microplate reader.

LDH was measured using a CytoTox-ONETM Homogeneous Membrane Integrity Assay Kit (Promega, Madison, WI, USA). Fluorescence intensity was measured at excitation and emission wavelengths of 560 and 590 nm, respectively. LDH release indicates potential cellular necrosis56 (link).
To establish the protective potentials of hepatoprotective drugs in hiHeps, hiHeps and PHHs were co-incubated for 24 hours with 4 hepatoprotective compounds (quercetin (40 μmol/L), silymarin (25 μmol/L), curcumin (15 μmol/L) and metformin (200 μmol/L)) and DCA in different assays at toxic concentrations (MTT assay: 1000 μmol/L; ATP assay: 500 μmol/L; MMP measurement: 1000 μmol/L; ROS assay: 1000 μmol/L; apoptosis assay: 200 μmol/L; and LDH release assay: 500 μmol/L).

Cell viability was measured using MTT and lactate dehydrogenase (LDH) release assays. MTT solution was added to the culture medium of cells (1 × 10⁵ cells/mL) (final concentration of MTT in the medium was 0.5 mg/mL), and the cells were incubated at 37 °C for 1 h (Lee et al. 2015 (link)). After removing the culture medium, 50 μL of DMSO (purity ≥ 99.9%) was added, and the optical density of each well was measured at 570 nm. LDH release assays were performed using a CytoTox-ONE^TM Homogeneous Membrane Integrity Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.