Visionblue quick cell viability fluorometric assay kit

To assay proliferation, we seeded MuSCs on flat hydrogels at a density of 500 cells per cm² surface area. We counted cell number using a hemocytometer. We collected cells at indicated timepoints by incubation with 0.5% trypsin in PBS for 5 min at 37 °C and quantified them using a hemocytometer at least 3 times. Additionally, we used the VisionBlue Quick Cell Viability Fluorometric Assay Kit (BioVision, catalog # K303) as a readout for cell growth in culture. Briefly, we incubated MuSCs with 10% VisionBlue in culture medium for 4 h, and measured fluorescence intensity on a fluorescence plate reader (Infinite M1000 PRO, Tecan) at Ex= 530–570 nm, Em=590-620 nm. Data analyses were blinded, where researchers performing cell scoring were unaware of the treatment condition given to sample groups analyzed.

To assess MuSC proliferation with different drug doses, we seeded 400 MuSCs isolated from young mice (2–4 mo.) on collagen coated 96 well plates in myogenic cell culture medium containing DMEM/F10 (50:50), 15% FBS, 2.5 ng ml⁻¹ fibroblast growth factor-2 and 1% penicillin-streptomycin. We performed 3-5 replicates per condition plated with 3 doses (1, 100 or 1000 nM). Each plate contained vehicle controls. Small molecules were added at a 2X concentration to achieve the final concentration. To avoid cell-washout effects, we did not change the medium throughout the assay. Proliferation was assayed at 7 days post-plating by using the VisionBlue Quick Cell Viability Fluorometric Assay Kit (BioVision, catalog # K303) as a readout for cell growth in culture. Briefly, we incubated live MuSCs with 10% VisionBlue in culture medium for 4 h, and measured fluorescence intensity on a fluorescence plate reader (Infinite M1000 PRO, Tecan) at Ex= 530–570 nm, Em=590-620 nm. Proliferation was normalized to the average value of vehicle treated for each plate. Data analyses were blinded, where researchers performing cell scoring were unaware of the treatment condition given to sample groups analyzed.

Cell proliferation was determined using a tetrazolium-based colorimetric assay (MTT), which measures only in vitro living cells and reveals the results related to the number of viable cultured cells. MTT (0.5 mg/mL) dissolved in PBS was added to the cells for 2 h of incubation at 37 °C. After removing MTT-containing medium, the produced purple formazan was dissolved in dimethyl sulfoxide and then measured at 570 nm against a 620-nm reference wavelength (ΔOD, OD: optical density) using a Multiskan microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA). Additionally, a trypan blue exclusion assay was applied to calculate the number of viable cells, which serves as a direct method to measure cell proliferation. Initially, cells were seeded into 24-well plates at a density of 1 × 10⁴ cells per well and exposed to an acute glucose shift the next day. At the indicated time points, cells were removed from the culture plates by trypsin-EDTA and stained with trypan blue for the cell count in Cellometer Auto T4 Cell Counter (Nexcelom Bioscience, Lawrence, MA, USA). For the cell viability assay, cell survival was determined by the VisionBlue™ Quick Cell Viability Fluorometric Assay Kit (BioVision, Mountain View, CA, USA) according to the manufacturer’s instructions. For details, please see Supplementary information.