4.2.3. MTT Viability Assay

Indirect estimation of relative cell numbers was conducted by the MTT colorimetry assay. In order to assess the sensitivity to DZNeP (3-Deazaneplanocin A HCl) (Selleck Chemicals LLC (Houston, TX, USA)), the MTT viability assay was performed. For each cell type, a single cell suspension solution of 50,000 cells/mL per well was seeded in a 24-well plate. The cells were treated with the methyl-transferase inhibitor 3-Deazaneplanocin A (DZNeP) (Selleck Chemicals LLC (Houston, TX, USA)) at a dose range of 0.05–40 μM, dissolved in DMSO (PanReac Applichem, ITW Reagents, Darmstadt, Germany). DMSO was used as vector to a final dilution of 1:1000. The plate was incubated up to 90–100% confluence. Followingly, the culture medium was removed from all wells and replaced with 200 μL MTT (M5655-1G, Merk, Sigma-Aldrich, Darmstadt, Germany) in a working solution of 1 mg/mL dissolved in PBS. The plate was incubated at 37 °C in darkness in CO₂ absence for ~3 h until the formation of intracellular purple formazan crystals. The crystals were solubilized in 300 μL of acidified isopropanol and the optical absorbance of the solution was monitored at 595 nm using a microplate spectrophotometer (ASYS HITECH, Biochrom, UK). Repeated measurements at 660 nm followed, for normalization over cell density. The dose-response curves were generated using the formula:

The control corresponds to the viable cell population measurements of the untreated wells.