Nbt bcip reagent kit

The cells used in the AlamarBlue ® proliferation evaluations were cultured in experimental medium for further 7 days. In particular, the concentrations of serum in the five experimental media E1-E5 were increased to 10%. The cells were then fixed using 4% paraformaldehyde (PFA) (Nacalai Tesque, Kyoto, Japan) phosphate buffered saline (PBS). After washing twice with 25 mM tris-buffered saline (TBS) (pH 7.4) (Bio-Rad Laboratories, Hercules, CA, USA) and further washing with 25 mM TBS (pH 10.0), ALP activity staining was performed using the NBT/BCIP reagent kit (Nacalai Tesque). Three wells per experimental group were stained in each group and presented one result from each group.

The hDPSC was seeded within a 48-well plate at a density of 12,000 cells/well. After 6 h seeding, the medium was changed to a cement-immersed medium and cells were grown for 7 days. The cells were then fixed in place using 4% paraformaldehyde (PFA) (Nacalai Tesque) phosphate buffered saline (PBS). After washing twice with 25 mM tris-buffered saline (TBS) with a pH of 7.4
(Bio-Rad Laboratories, Hercules, CA, USA) and further washing with 25 mM TBS with a pH of 10.0, ALP activity staining was performed using the NBT/BCIP reagent kit (Nacalai Tesque).