Celltiter 96 aqueous one solution cell proliferation assay mts test

Cell survival was measured using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS test, Promega, USA). Cells have been seeded in a 96-well flat-bottom plate in 100 μl of culture medium (10³ cells/well for A549, HT-1080, dermal fibroblasts and eMSC or 5 × 10³ cells/well for ZR-75-1) and allowed to attach for 24 h. The starting amount of cells was chosen to account for their growth rate and degree of spreading such as to avoid excessive dilution in the beginning of the experiment or control cell overgrowth toward the end. After 24 h, 10 μl of peptide stock solution or PBS (control) were added to the wells. After 96 h, 20 μl of the MTS-reagent were added to each well and 1–2 h later optical density was measured at the wavelength 492 nm using the 96-well plate reader (Titertek Multiskan, Labsystems, Finland). All experiments were conducted in duplicate or triplicate. The same protocol has been used with the other treatment schemes using multiple additions of the peptide, see Fig. 2, or different incubation times, see Fig. 9.

Cell viability was determined using the CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS test, Promega, Charbonnières-les-Bains, France). Cells were seeded in 96-well plates at a density of 2.10³ for 24 h before treatment with 0 to 100 µM of WN198, WN191, WN170, WN197, or cisplatin for 72 h. Cells were incubated for 2 h with 20 µL of CellTiter solution at 37 °C in 5% CO₂, and the production of formazan from reduced 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was measured at 490 nm (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany). GraphPad Prism V6.0 software served to calculate IC₅₀. Statistical differences between WN198 and WN191 were ascertained by a Student’s t-test (* p < 0.05, ** p < 0.001, *** p < 0.0005 and **** p < 0.0001).