Analysis of Cell Viability by MTT Assay

Cells were seeded into a 96-well plate and incubated at 37°C (5% CO₂) for 24 h. After attachment, primary microglial cells were treated according to the stimulation scheme depicted in Figure 1A. Twenty-four hours after the second stimulation by LPS, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to primed cells and incubated for 4 h at 37°C (5% CO₂). In parallel, the same procedure was performed on US cells as well as on UP cells (stimulation occurred solely on day 6 with a fixed dose of 100 ng/ml LPS). Afterwards, the solubilization solution was added to each well and incubated overnight at 37°C (5% CO₂) for 24 h. Absorbance was measured at 570 nm using a TECAN Infinite 200 Plate Reader (Tecan, Switzerland).