Evaluating Cell Viability with AdipoRon

Cell viability was determined by 3-[4.5-dimethylthiazol-2-yl]-2.5-dipheniltetrazolium bromide (MTT) colorimetric assay according to the manufacturer’s instructions. Briefly, U-87 and SH-SY5Y cells were seeded in 96-well plates (2 × 10³/well) and incubated overnight in DMEM 10% FBS medium. The day after, the cells were treated with increasing doses of AdipoRon (2.5, 5, and 10 μg/mL) (Sigma-Aldrich, MO, USA), or treated with MS-CSF from MS patients (10%) or co-treated with AdipoRon (2.5 μg/mL) and MS-CSF (10%). As control, SH-SY5Y and U-87 cells were incubated in 5% FBS medium alone. After 24, 48, and 72 h of treatment, cells were stained with MTT reagent and processed as previously described [24 (link)]. Each experiment was performed two times in triplicate for MS-CSF and AdipoRon treatment alone and three times for the combined treatments.