In vitro cytotoxicity

A colorimetric 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazodium bromide (MTT) viability assay³⁸ was used to assess the in vitro cytotoxicity of LTX-315, as well as a selection of cytostatic drugs against the rTMSC cell line. Pre-cultured cells were transferred to a 96-well plate in subconfluent concentrations at a volume of 100 μl/well (culture media). Cells were left overnight in a cell incubator under conditions of 37 °C; >95% humidity and 5% CO₂. Wells were plated in triplicate. LTX-315 and the different cytostatic drugs were dissolved in a serum-free RPMI 1640 and diluted to a concentration range of 7–100 μM and 10–100 μM, respectively. The cytostatic drugs were originally dissolved in dimethyl sulfoxide (DMSO) and later in a serum-free RPMI 1640, yielding a final DMSO concentration ranging from 0.5% in the 100 μM solution to 0.05% in the 10 μM solution. Cells were washed once with serum-free RPMI 1640 before incubation with the peptide solutions or cytostatic drugs for 2, 4, 8, 24 and 48 h, respectively. Cells in a serum-free media alone were used as a negative control, while cells treated with 1% Triton X-100 (Sigma-Aldrich) in a serum-free media were positive controls. A 10 μl MTT solution (5 mg MTT per ml phosphate buffered saline) was added to each well, and the incubation was continued for an additional 2 h. Seventy microliters of solution were removed from each well, and 100 μl of 0.04 M HCl in isopropanol was added before the plates were shaken in an orbital shaker for 1 h at room temperature to help facilitate formazan crystal solubilization. The absorbance was measured at 590 nm on a microtiter plate reader (Thermomax Molecular Devices). The percentage of viability was calculated as the A590 nm of peptide-treated cells relative to the negative control (100% living cells), and expressed as a 50% inhibitory concentration (IC₅₀).