Sigmafast bcip nbt assay

hMSCs were seeded on the HA hydrogel array at a density of 20,000 cells/cm² and allowed to grow in expansion medium for 1 day. Cells were then shifted to a mixed differentiation medium, consisting of a 1:1 mixture of osteogenic and adipogenic differentiation media (StemPro kits, Thermo Fisher Scientific). Cells were maintained for 7 days in these conditions with media change every 3–4 days.
Adipogenic differentiation of PFA-fixed hMSCs was assessed by lipid droplet staining of BODIPY™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene, Invitrogen), at a 2 μM working concentration. The cells were then washed extensively with PBS prior to imaging on a Nikon Eclipse Ti-E epifluorescence microscope. After fixation and permeabilization, osteogenic differentiation of cells was assessed by alkaline phosphatase activity using the SigmaFast BCIP/NBT assay (Sigma-Aldrich). A working solution of BCIP/NBT was prepared as per manufacturer’s instructions, added to the cells, incubated for 15 min at room temperature, and then washed extensively in 1X PBS. Alkaline phosphatase staining was identified as a dark purple color and imaged on an Olympus IX50 inverted fluorescence phase contrast microscope, with images captured by a Canon EOS Rebel T3 digital SLR camera.