Quantifying Alkaline Phosphatase Activity

To assess ALP activity, cells were washed twice in PBS, stained with BCIP/NBT Blue Liquid Substrate solution (B3804 Sigma-Aldrich), and incubated in the dark for 10 min at room temperature. The colorimetric reaction was blocked by washing twice with distilled water [50 (link)]. The intensity of ALP staining was assessed under an Evos bright-field microscope (EVOS M5000 Cell Imaging System, Life Technologies). The images were quantified by an ImageJ-based method [36 (link)] to evaluate blue staining in the RGB 24-bit acquisition. The number of blue pixels with higher intensity than the red or green pixels was divided by the total pixels in the entire field for each of the three independent replicates.