ALP activity and staining were carried out to study the osteogenic potential of Murine preosteoblast MC3T3-E1 cells under experimental conditions, according to our previously published protocol (Behera et al., 2018 (link)) and others (Ming et al., 2013 (link); Y. K. Zhai et al., 2014 (link)). Briefly, MC3T3-E1 cells were grown in an osteogenic medium in 12-well culture plates. After 9 days of osteogenic induction, the cells were fixed with 4% formaldehyde and stained with SIGMAFAST™ p-nitrophenyl phosphate tablets (catalog No. N1891; Sigma-Aldrich, St. Louis, MO), and the images were then photographed. The number, total area, and relative intensities of ALP-positive colonies were counted and analyzed by using the image analysis software Image-Pro® plus 6.0.