Versamax enzyme linked immunosorbent assay elisa microplate reader

The 3-(4,5-dimethylyhiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was carried out to analyze the effect of VCA on the proliferation ability of PD-MSCs and BM-MSCs. PD-MSCs and BM-MSCs were seeded at 2.0 × 10³ cells per well on a 96-well plate (Thermo Scientific, Waltham, MA, USA) and cultivated at 37 °C in an incubator with a humidified atmosphere of 5% CO₂. After 24 h, the cells were treated with VCA (0–10,000 pg/mL) for 48 h; 0.5 mg/mL MTT (Sigma) was added to the VCA-treated cells for 3 h, and then 200 µL of dimethyl sulfoxide (DMSO) (Sigma) was added for 10 min. The resulting formazan was dissolved in DMSO, and absorbance was detected at 570 nm with a VersaMax enzyme-linked immunosorbent assay (ELISA) microplate reader (Molecular Devices, San Jose, CA, USA). Experiments were performed in triplicate.

Total phenol content of the sample was measured according to the method described by Chandra et al. [38 ] with some modifications. Briefly, 20 μL of each fraction (at concentration of 1000 μg/1 mL methanol) was mixed with 50% Folin–Ciocalteu phenol reagent (20 μL) in 96-well plates. After 5 min, 1 N sodium carbonate solution (20 μL) was added to the mixture and distilled water was added to adjust the final volume to 200 μL. After incubation at room temperature (RT) in the dark for 30 min, the absorbance of test sample against a blank was measured at 725 nm using a VersaMax enzyme-linked immunosorbent assay (ELISA) microplate reader (Molecular Devices, LLC, San Jose, CA, USA). Total phenol content was calculated based on a calibration curve of gallic acid. The results were expressed as mg gallic acid equivalent (mg GAE)/g.