Quantitative Analysis of Osteogenic Mineralization

Mineralization was induced on confluent monolayers in 12-well plates by addition of osteogenic media. Monolayers were washed with 1X PBS and fixed for 1 h with cold ethanol (70%, v/v), then washed 3 times with excess dH₂O prior to addition of 1 mL of Alizarin Red (2% w/v, pH 4.2) per well. The plates were incubated in the dark at RT for 10 min. After removal of unincorporated dye, wells were washed three times with dH₂O, re-aspirated, and stored at room temperature (RT). In identical cultures for the mineralization assay, monolayers of MPC1 and MPC2 cells were washed twice with 1X PBS and fixed for 30 min in 4% PFA. PFA was removed and cells were washed again in 1X PBS, then stained using the Leukocyte Alkaline Phosphatase Kit (Sigma) as previously described^{18 (link)}. Plates were stored at RT. Images were taken at 10X magnification on an inverted Leica microscope. Alizarin red staining was quantified using cetylpyridinium chloride (CPC) extraction for 3 h as previously described^{19 (link)} and absorbance was measured at 544 nm. Intensity of alkaline phosphatase staining was quantified using ImageJ, as previously described^{20 (link)}.