Alizarin red s staining kit

Before the analysis of osteogenic properties, cells were treated with osteogenic induction medium consisting of DMEM with 10% FBS, 25 mg/ml ascorbate-2 phosphate, 10⁻⁸ M dexamethasone, and 5 mM β-glycerophosphate (All from Gibco, USA) for the indicated time (otherwise 2 weeks). After induction, cells were fixed with 4% paraformaldehyde, after few washes with PBS, cells were stained according to the manufacturer’s instruction. We used Alizarin Red S Staining Kit (#0223, Sciencell, San Diego, USA) for calcium deposition analysis, Alizarin Red S Staining Quantification Assay kit (#8678, Sciencell, San Diego, USA) was used for quantification. Alkaline phosphatase staining kit (1102-100, Sidansai, Shanghai, China) was used for cellular ALP staining, and Alkaline Phosphatase Assay kit (ab83369, Abcam, Beverly, USA) for quantification. The average value of OD_405nm of each sample were calculated, and the average value of OD_570nm of each sample were also calculated as background control. Values of OD_405nm were divided by OD_570nm to generate a standardized OD value for each sample for further comparison. Infinite™ M200 (Tecan, USA) microplate plate reader was used for absorbance quantification, and three replicates were done to generate the average value.

To verify the stemness of rMSCs^HIF-2α, we determined the expression of cell surface CD29 and CD45 by flow cytometry and tested the multiple differentiation potential of rMSCs^HIF-2α. For flow cytometry, rMSCs^HIF-2α were harvested, incubated with R-phycoerythrin (PE) hamster anti-rat CD29 (1 : 100, 562154, BD Biosciences, San Jose, CA, USA) and PE mouse anti-rat CD45 (1 : 50, sc-70696, Santa Cruz Biotechnology Inc., Dallas, TX, USA) for one hour, and analyzed on a flow cytometer (BD LSR II Flow Cytometer System; BD Biosciences, San Jose, USA). RMSCs^HIF-2α were induced to differentiate into adipocytes, osteoblasts, or chondrocytes by incubating with adipocyte, osteoblast, or chondrocyte differentiation induction medium (Cyagen Biosciences Inc., USA). Lipid expression and production in the differentiated fat cells were determined by oil red O staining (Oil Red O kit was from BioVision Inc., Milpitas, CA, USA) according to the protocol from the kit. Calcium deposit in the differentiated osteoblasts was stained with alizarin red S according to the protocol in the alizarin red S staining kit (Sciencell Research laboratories, San Diego, CA, USA). Glycosaminoglycan matrix in the differentiated chondrocytes was stained with toluidine blue (toluidine blue stain kit was from GeneCopoeia Inc., Rockville, MD, USA) according to the protocol provided by the manufacturer.

Cultured cells were stained by Alizarin Red S staining kit (ScienCell Research Laboratories) as per manufacturer’s protocol. Briefly, the media were carefully removed from the cultured cell wells and cells were washed three times with PBS. Cells were fixed with 4% formaldehyde/PBS for 15 min at room temperature. The fixative solution was removed and cells were washed three times with deionized water. The water was removed and 500 μl of 40 mM Alizarin Red S dye was added per well. Cells were incubated at room temperature for 30 min with gentle shaking. The dye was removed and cells were washed five times with deionized water before taking images. For tissue transplants, the sections were deparaffinized and hydrated to 70% ethanol. Then, the tissue sections were fixed with 10% formaldehyde/PBS for 15 min at room temperature before staining following the protocol described above.

On day 21 of osteoblast differentiation, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The fixative was rinsed, and the cells were then washed 3 times with distilled water and stained with the 2% Alizarin Red S Staining Kit (ScienCell Research Laboratories, Cat. No. 0223) for 10–20 min at room temperature. Later, the cells were washed with water and images were taken under the microscope.

The cell layer was washed with PBS, and then fixed with 4% paraformaldehyde for 15 min at room temperature. After removing the fixative, the cell layer was rinsed in distilled water and stained with the 2% Alizarin Red S Staining Kit (ScienceCell, Research Laboratories, Cat. No. 0223) for 20–30 min at room temperature. Excess dye was washed off with water. For quantifying the Alizarin Red S staining, the Alizarin Red S dye was eluted in 800 μl of acetic acid incubated in each well for 30 min at room temperature as described^{59 (link)} and measured in spectrophotometer (BioTek, Epoch) at 405 nm.