Alizarin red ph 4.2

The preconditioned PDLSCs were cultured in 6-well plates with osteogenic inductive medium. Twenty-eight days later, the cells were washed three times with PBS and fixed in 4% paraformaldehyde for 15 min. Then, the cells were washed for another three times and stained with Alizarin red (PH 4.2; Sigma-Aldrich) for 5 min. Then, the formation of mineralized nodule was observed under inverted microscope.

The detection of the mineralized matrix was evaluated at the end of the 17 days of the experiment. After removing the culture medium, the wells were washed three times with PBS (GibcoTM, USA) heated to 37 ºC, and 2 mL of 10% formalin was added for fixation and maintained at 4 °C for 24 hours. After that time, formalin was removed and the wells were dehydrated at room temperature in increasing series of alcohols (30%, 50%, 70%, and 100%) for a period of 1 hour for each alcoholic graduation. After drying, the wells were stained with 2% alizarin red pH 4.2 (Sigma) for 10 minutes, and the mineralized areas rich in calcium were evidenced by the red color. The quantification was performed according to Gregory et al. ¹², where 280 μL of 10% acetic acid (Labsynth, Brazil) were added to each well and the plates were left under gentle agitation for 30 minutes. The cell layer was then scraped with the aid of a tip and the solution was transferred to 1.5 mL Eppendorf tubes, heated to 85 ºC for 10 minutes, and transferred to the ice for 5 minutes. The tubes were centrifuged at 13.000 rpm for 20 minutes. A volume of 150 μL of the supernatant was transferred to a 96-well plate (Corning) and 40 μL of 10% ammonium hydroxide (Quimibras, Brazil). The reading was performed on a spectrophotometer (Bio-Tek) at a wavelength of 405 nm.

For osteogenic differentiation, adMSC were seeded in 6-well plates at a density of 1 × 10³ cells/well and cultured until subconfluency. Then, the culture medium was replaced with medium consisting of α-MEM, 10% FBS, supplemented with 50 μg/mL l-ascorbic acid 2-phosphate (Sigma), 10^–8 M dexamethasone (Sigma) and 10 mM β-glycerophosphate (Sigma) in order to induce the deposition of mineralized matrix. After 21 days, cells were fixed in 3.7% paraformaldehyde for 20 min and stained with 1% Alizarin Red (pH 4.2) (Sigma) for one hour at room temperature.
For adipogenic differentiation, adMSC were seeded in 6-well plates at a density of 1 × 10³ cells/well and cultured until subconfluency. Then the culture medium was replaced with complete medium supplemented with dexamethasone (0.5 μM), indomethacin (50 μM) (Sigma) and isobutylmethylxanthine (0.5 mM) (Sigma). After 14 days, to detect lipid accumulation, cultures were fixed in 3.7% paraformaldehyde for 20 min and stained with 0.3% Oil Red O in isopropanol for one hour at room temperature.