Sigmafast p nitrophenyl phosphate kit

For microscopic views, we used the BCIP/NBT kit (Sigma) to detect alkaline phosphatase activity. Cells were washed once with Ca⁺⁺ and Mg⁺⁺-free PBS containing 0.05% Tween 20, then fixed with 10% formalin for 1 minute, and stained with BCIP/NBT substrate in the dark for 5–10 minutes. Afterwards, cells were washed once with the wash buffer, and pictures were taken. Positive staining is a dark blue color. To quantify the relative alkaline phosphatase activity, we used the SIGMAFAST™ p-Nitrophenyl Phosphate kit (Sigma) for AP measurement and the Thiazolyl Blue Tetrazolium Blue (MTT) assay (Sigma) for cell number.

After 3 or 7 days in culture cell viability (proliferation) was assessed
with the colorimetric MTS assay (Promega, Madison, WI, USA) (Cory et al., 1991 (link)). Briefly, 20 μL
of the MTS solution was added to each well and incubated at 37 °C for 1
hour. The 96 well plate was read at 490 nm on a Gen5 plate reader for absorbance
(BioTek, Winooski, VT, USA). Quantitative ALP activity was assessed at 3 and 7
days. Cells were lysed in a 0.1% Triton X lysis buffer and whole cell lysate was
measured using SigmaFast p-Nitrophenyl phosphate kit (Sigma-Aldrich, St. Louis,
MO, USA) by adding para-nitrophenylphosphate (pNPP) as a substrate assay buffer
containing MgCl₂ for 30 minutes, and the kinetic of absorbance was
read at 405 nm. Standard t-test and ANOVA with post-hoc Bonferonni analyses were
conducted on quantitative data (MTS, ALP) where appropriate. Violations of
homogeneity of variation resulted in the use of Welch's correction. A total of
n=8 wild-type control cell populations were used in the bioassays. For Twist
+/− we had n=8 for MTS but some cell population loss for our ALP assay
resulting in an n=5 for 3day and n=7 for our 7day Twist +/− data.