4.3. In Vitro Cytotoxicity of MTX and GLU–MTX

For MTT experiments, cell lines were seeded in 96-well plates (5−8 × 103 cells/well). The following day cells were treated with an appropriate complete culture medium (control) and different doses of methotrexate and glucose conjugated MTX (10, 50 µM) for 48 h.

After the incubation, an MTT assay was performed. Cell viability was evaluated by the conversion of the yellow tetrazolium salt (MTT) into violet formazan insoluble crystals in mitochondria of active cells. Following 4 h, the medium was removed, and the dye was dissolved by dimethyl sulfoxide (DMSO, Sigma-Aldrich, Munich Germany), creating the color, which intensity is proportional to the viable cells. The absorbance rate was measured at 490 nm, and the reference wavelength was 570 nm (Bio-TekBioTek ELX800 multi-well reader, BioTek, Winooski, VT, USA). The viable cells (VC) were calculated as VC (100%) = (absorbance of experimental group/absorbance of the control group) × 100%. MTT experiments were repeated, and figures represent the mean with standard deviation.