Cytotox one homogeneous membrane integrity assay reagent

MCF-7 cells were seeded in 96 well microplates at a concentration of 5 × 10³ cells/well and stabilized in an incubator for 24 h. Successively, TiO₂NPs at two different concentrations (25 and 50 µg/mL) were added to cell media. After incubation times of 24 and 48 h, standard WST-8 assay (96992, Sigma Aldrich, Darmstadt, Germania) and the lactate dehydrogenase (LDH) leakage assay, using the CytoTox-ONE Homogeneous Membrane Integrity Assay reagent (G7890, Promega, Madison, WI, USA), were performed to conduct viability testing and to evaluate the membrane damage, respectively.
The WST-8 assay procedure that was used is described in our previous work [43 (link)].
The amount of lactate dehydrogenase (LDH), a soluble cytosolic enzyme released after cell lysis, was measured by reading absorbance at 490 nm using a Bio-Rad microplate spectrophotometer. The increase of the LDH activity in culture supernatant is proportional to the number of cells lysed. Data were expressed as mean ±SD. Mean values differences between cells treated and respective controls were considered statistically significant performing a t-student test (p-value < 0.05)

Cytotoxicity was evaluated using the CytoTox ONE™ Homogeneous Membrane Integrity Assay reagent (Promega, WI, USA), which measures LDH release. HaCaTs were grown on different scaffolds for one, three, or five days and culture media was exchanged every two days. At each incubation time point, plates were first incubated at room temperature for 20 min, and the supernatant was collected and mixed with an equal volume of LDH assay reagent mix according to manufacturer instructions. After 15 min of incubation, the reaction was stopped, and fluorescence was measured at 560 nm/590 nm using an Agilent BioTek Synergy H1 hybrid multi-mode fluorescence microplate reader. The LDH positive control was used to lyse cells and obtain the maximum possible LDH release.

Membrane damage in HeLa, MCF-7, and Caco-2 cells exposed to increasing doses of PdNPs (25, 50, and 100 μg/mL), was obtained by quantifying the release of lactate dehydrogenase (LDH), a product of lipid peroxidation. HeLa (5000 cells/well), MCF-7 (5000 cells/well), and Caco-2 (50000 cells/well) cells were seeded in a tissue culture-treated 96-well plate (Constar) containing 100 μL of complete DMEM and incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 24 h. DMEM was then replaced with fresh medium containing PdNPs and cells were incubated for 24, 48, and 72 h. Afterwards, the LDH assay was performed by using the CytoTox-ONE homogeneous Membrane Integrity Assay reagent (Promega Corporation, Madison, WI, USA). Results were recorded by using an Infinite 200 Pro plate reader. Data were normalized with respect to cells treated with lysis buffer in the same conditions (positive control, P, expressed as 100%). Results are reported as mean ± SD.