2.7. Cell Viability Assay (MTT)

The cytotoxicity of mRNA modifications was assessed using the MTT test [26]. HEK293FT, PC3, and A549 cells (1 × 105 cells/mL) in 100 μL cell culture were seeded in 96-well plates and incubated by night. The next day, mRNA-M0, mRNA-M2, mRNA-M6, and mRNA-M7 were added to the cells at a concentration in the range from 50 μg/mL to 0.4 μg/mL and incubated for 72 h. After 72 h of cell incubation with the tested complexes, 20 μL of the MTT solution (5 mg/mL) was added to each well and incubated for 2 h in a CO₂-incubator. After incubation, we removed the medium and added dimethyl sulfoxide (50 μL/well) to terminate the reaction. Optical density was detected via a microplate reader Varioskan LUX (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 570 nm. Cell viability in the presence of complexes was calculated as follows: [(optical density of texted wells/optical density of control wells) × 100%].