Microplate manager software 6 version 6

Parental clone #2Luc/GFP and #2LucRi/GFP MSCs were seeded 1.5 × 10⁴ cells/well in 0.5 ml of complete medium in 48-multi-well plates. After 24, 48, 72, and 96 h from cell seeding, culture medium was replaced with 0.2 ml of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) powder (0.5 mg/ml thiazolyl blue tetrazolium bromide, Sigma) diluted in serum-free culture medium. Plates were then incubated for 4 h at 37°C. The deposition of violet crystals in the bottom well reveals the presence of metabolic active cells. The solution was then removed and crystals were dissolved in 0.2 ml of dimethyl sulfoxide (DMSO). Absorbance was immediately quantified using a plate reader (xMark Microplate Spectrophotometer, Bio-Rad) at 570 nm wavelength, and optical densities were quantified through the Microplate Manager Software 6 version 6.3 (Bio-Rad). At each time point of observation, the proliferation assay was measured in triplicate for both cell lines, and the assay was repeated at least twice with similar results.

Cells were seeded (5 × 10⁴ cells/well) in 0.5 mL of complete medium in 48 multiwell plates. After treatment, the supernatants were replaced with 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT solution, Sigma-Aldrich) diluted in serum-free culture medium. Plates were then incubated for 2 h at 37°C. The deposition of violet crystals in the bottom well reveals the presence of metabolic active and vital cells. The solution was then removed, and crystals were dissolved in 0.2 mL of dimethyl sulfoxide (DMSO, Sigma-Aldrich). Absorbance was immediately quantified using a plate reader (xMark Microplate Spectrophotometer, Bio-Rad) at 570 nm wavelength and optical densities (O.D.) were quantified through the Microplate Manager Software 6 version 6.3 (Bio-Rad). At each time point of observation, the viability was measured in triplicate for both the cell lines, and biological replicates were performed at least twice with similar results. Results were expressed as % of vital cells compared with untreated cells as control (considered as 100% by default) at each time point.