Viability and EdU proliferation assay

SCs were seeded at a density of 4000 cells/well in 384-well microplate (Greiner) for overnight incubation in complete culture media. The following day, the media was replaced by serum-free media with increasing concentrations of human VEGF recombinant protein, mouse PDGF-BB recombinant protein from 0 to 100 ng/mL, as single and combined dose. After 20 h of incubation, 5 µL of resazurin (500 µM, Sigma) was added to each well to reach to a final concentration of 50 µM. Following this, the plate was incubated for further 4 h at 37 °C and 5% carbon dioxide (CO₂). The fluorescent signal of the metabolized component, resorufin, was then quantified using POLARstar Omega (BMG Labtech) plate reader at (584/590-10). For EdU proliferation assay, Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 dye (Thermofisher) was used, and manufacturer’s protocol was followed with slight modification. Similar conditions were maintained, as mentioned previously, with addition of EdU labelling solution (2 mM) for 24 h. The plate was then fixed and stained with p75NTR antibody to identify EdU and p75NTR positive cells. Images were acquired using Nikon Ti2 widefield microscope and approximately 160 cells (~ 60% p75 positive cells) were counted from each field of view (FOV). A total of 36 FOV were used per technical replicate and further processing was done with Cell Profiler 4.2.1 software. Representative images were taken using Olympus FV3000 confocal microscopy at 20X objective.