2.6. Alizarin Red Staining

Extracellular calcium deposits of osteo-induced hBM-MSCs were observed by Alizarin Red staining. Cells were cultured in 6-well plates (1 × 10⁵ cells/well) and osteo-induced as previously described. At day 14 of differentiation, hBM-MSC osteoblasts were fixed on wells by addition of 1 ml ice-cold 70% EtOH at 4°C after aspirating the medium and washing wells with PBS. After 1 h, EtOH was removed from wells and cells were washed with distilled water. Alizarin Red S (A5533, Sigma-Aldrich) dyeing solution (pH 4.2, 2% w/v in distilled water) was then added to each well (1 ml/well), and the plates were kept at room temperature for 10 min in the dark. After staining, wells were aspirated, and cells were washed four times with 1 ml distilled water. Images of cells were taken by an Olympus microscope (Tokyo, Japan). Subsequently, the Alizarin Red dye was eluted from wells with 10% (m/v) cetylpyridinium chloride (C0732, Sigma-Aldrich) in 10 mM sodium phosphate buffer (P5244, Sigma-Aldrich) solution and mineralization was quantified by absorbance values at 560 nm using a Multiskan GO microplate reader (Tecan Austria GmbH).