Bio vision alkaline phosphatase activity colorimetric assay kit

Alkaline phosphatase was measured using the Bio Vision Alkaline Phosphatase Activity Colorimetric Assay Kit (K412‐500; BioVision). All buffers and components used for the assay were supplied in the kit. For the collection of cells for the ALP assay, cells were initially washed with 1× PBS and the plates with the washed cells were stored at −80°C. On the day of the assay, frozen cells were taken up in Alkaline Phosphatase Buffer and homogenized by pipetting up and down. A p‐nitrophenyl phosphate (pNPP) substrate tablet was taken up in 2.7 ml Alkaline Phosphatase Buffer. A total of 80 µl of each sample was pipetted into each of four wells of a 96‐well plate. At least three control wells with buffer only were included on each plate. After all the samples were pipetted, 50 µl of the pNPP substrate was added. The samples were incubated at room temperature in the dark for 60 min. Stop Solution (20 μl) was added to each well at the end of the incubation time. The absorbance was measured using the Synergy 2 BioTek multipurpose microplate reader at 405 nm.

To measure TNAP activity, cells or ultracentrifugation pellets were re-suspended in 120 µl of TNAP Assay Buffer (BioVision) and incubated overnight at −30°C. TNAP activity was measured using the BioVision Alkaline Phosphatase Activity Colorimetric Assay kit according to the manufacturer's protocol (BioVision).