Sigmafast bcip nbt detection kit

HAT-7 cells were cultured within oxidized alginate-carboxymethyl chitosan hydrogels for 14 days and then stained with an alkaline phosphatase (ALP) staining kit to detect the expression of alkaline phosphatase, a marker of ameloblast differentiation. Cell-laden constructs were rinsed with PBS twice and cells encapsulated in the hydrogels were fixed and permeabilized using BD Cytofix/cytoperm^TM fixation/permeabilization kit (BD Biosciences, San Jose, CA, USA) by incubating the constructs in the fixation and permeabilization solution for 30 min at 4 $℃$ . Cell-encapsulated hydrogels were washed with 1 $\times$ BD Perm/Wash^TM buffer and then with PBS, twice each. Then, alkaline phosphatase expression in cell-laden constructs was detected using SIGMAFAST^TM BCIP^®/NBT detection kit (Sigma-Aldrich). The ALP staining reagent was prepared by dissolving 1 tablet from SIGMAFAST^TM BCIP^®/NBT detection kit (Sigma-Aldrich) in 10 mL of distilled water as instructed by the manufacturer. Cell-laden constructs were incubated in ALP staining reagent at 37 °C under dark condition for 24 h. Cell-encapsulated hydrogels were then visualized under a light microscope (EVOS M5000 Cell Imaging System, Thermo Fisher Scientific).