Mts based colorimetric assay

Prior to the treatment with S. aureus and LPS the cytotoxic effect was studied using a MTS-based colorimetric assay (Promega Corporation, Madison, WI, USA) in which mitochondrial activity in viable cells transforms tetrazolium compound into colored formazan. A total of 17 000 cells/well were seeded into 96-well plates in a volume of 100 μl, cultured and differentiated as described above. Cells were tested with 100 μl of either S. aureus suspensions (1 x 10⁴, 10⁵, 10⁶, 10⁷ 10⁸ CFU/ml) or vehicle controls as described above. After incubation with S. aureus or LPS, 20 μl CellTiter 96^® AQ_ueos One Solution Reagent was added to each well, according to MTS manufacturer’s instructions. After 1 hour of incubation at 37°C absorbance was measured at 490 nm using a Wallac Victor²1420 microplate reader (Perkin-Elmer, Massachusetts, USA). Cell viability was assessed by comparing mean absorbance values from bacteria or LPS treated cells and vehicle controls based on six replicates. Six wells were used per concentration and for controls.

HFFs (1 × 10⁴ cells/well) were seeded in 96-well plates and then treated with various concentrations of Eltanexor as indicated and incubated at 37°C for 72 h. Eltanexor was dissolved in dimethyl sulfoxide (DMSO), and an equal volume of DMSO served as a vehicle control. Cell viability was determined by a 3-(4, 5, dimethyliazol-2-yl)-5-(3carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS)-based colorimetric assay (Promega) as described by manufacturer’s instructions (Meloni et al., 2015 (link)). 50% cytotoxic concentration (CC₅₀) is determined according to non-linear trajectory analysis using GraphPad Prism Software (San Diego, CA).