Alizarin Red S staining and assay

The medium from samples (n = 6) was removed and samples were gently washed three times in 1X PBS. Cells were fixed using cold 70% ethanol for 1 h and allowed to air dry. A 0.5% ammonium hydroxide (Sigma-Aldrich, MO) solution was prepared by diluting 30% stock solution in distilled water. Alizarin Red S (ARS) (Sigma-Aldrich, MO) staining solution (40 mM) was prepared by dissolving the dye in distilled water. The pH was adjusted to 4.1–4.3 using 0.5% ammonium hydroxide. The samples were incubated in ARS solution for 1 h at room temperature, followed by three distilled water rinses and imaging. A 10% cetylpyridinium chloride (Sigma-Aldrich, MO) solution (w/v) was made by dissolving in 0.1 M sodium phosphate buffer (pH 7.0). The ARS samples were destained in this solution and the absorbance readings (A.U.) were obtained at 562 nm.