2.3. Analysis of Cell Viability

One day before the experiment, HMC3 cells were harvested by using a trypsin-EDTA solution, counted with a C-Chip disposable hemocytometer, and plated in 96-well plates (8 × 10³ cells/well). The following day, cells were treated with increasing concentrations of carnosine (1, 10, and 20 mM) and incubated for 24 h in a humidified environment (37 °C, 5% CO₂). Cell viability was measured through the well-known 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) method [34]. Briefly, after the stimulation process, MTT solution (1 mg/mL in EMEM medium) was added to each well and cells were incubated (2 h, 37 °C). During the final step, dimethyl sulfoxide was used to melt the crystals, while the Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek, Shoreline, WA, USA) was used to read the absorbance at 569 nm. Data are the mean of four independent experiments (n = 4). Values were normalized with respect to control untreated HMC3 cells and are expressed as the percent variation of cell viability.