*4.4. MTT Test*

To determine the cytotoxicity of glucose and insulin treatment on neuron-like cells, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay was performed according to the protocol of Liu et al. [59] with some modifications. This colorimetric assay is based on the ability of succinate dehydrogenase to reduce yellow MTT tetrazolium salt into blue MTT formazan crystals in living cells. The level of conversion provides an indication of mitochondrial metabolic function. Then, 5 × 103 neuron-like cells were incubated with diverse concentrations of glucose or insulin for 24 h in a CO2-incubator (37 ◦C, 5% CO2 and 95% humidity). Next, the supernatants were removed, and the cells were washed 3 times with 100 μL of PBS to remove phenol red residue, and 100 μL of 0.5 mg/mL MTT solution in PBS was added. The cells were incubated for 4 h at 37 ◦C in 5% CO2 and 95% humidity in the darkness. Subsequently, the MTT solution was carefully discarded, the formazan crystals were dissolved in 100 μL DMSO acidified with 1N HCl, and plates were shacked for 30 min at room temperature. Cell viability was determined by measuring the absorbance at 570 nm on the Synergie multiwell scanning spectrophotometer (BIOKOM, Janki, Poland). Cell viability for each glucose or insulin concentration was calculated as the percentage of the untreated neuron-like cells. All experiments were performed on 5 wells per concentration and repeated at least 3 times.
