Alizarin red s stain ars

Cell culture-grade chemicals were used in this study. Low glucose Dulbecco’s Modified Eagle’s Medium (LG-DMEM) and FBS were acquired from Bio West, Nuaillé, France. Penicillin–streptomycin, Amphotericin-B, and trypsin-EDTA (0.5% and 5.3 mM w/v, respectively) were obtained from Caisson, Smithfield, UT, USA. Minimum essential medium-alpha (α-MEM) was purchased from Gibco, Carlsbad, CA, USA, and Ex-Cyte from Millipore, Billerica, MA, USA. Insulin (3.5 mg [100 IU]/mL) was obtained from Novo Nordisk, Søborg, Denmark. MTT dye, collagenase type I, and TRIZOL (TriQuick, Catalogue #R1100) reagent were obtained from Solarbio, Fengtai, China. Dimethyl sulfoxide (DMSO), formalin, Triton X–100, isobutylmethylxanthine (IBMX), Dulbecco phosphate buffer saline (DPBS^−/−; without Ca²⁺ and Mg²⁺), and Alizarin Red S stain (ARS) were procured from Sigma-Aldrich, Taufkirchen, Germany. The antifade mounting media was obtained from Vecta Shield, St. Neots, UK. The cDNA synthesis kit (Catalogue # cDSK01-100) was purchased from Vivantis Technologies, Selangor, Malaysia. SYBR green master mix and Oil red O (ORO) were obtained from Thermo Scientific, Chino, CA, USA. T-25, and T-75 cell culture flasks, serological pipettes, 6-well, 24-well, and 48-well cell culture plates, and cell strainers were received from Corning, NY, USA.

Alizarin red S stain (ARS) (Sigma-aldrich, Milan, Italy) solution was prepared as described in [10 (link)].
HA-Si scaffolds were incubated with CGF pieces in a 12-well culture plate in BM or OM for 21 days. As a control (CTR), undifferentiated primary cells released from CGF were seeded in a 12-well culture plate in BM. Culture medium was changed at a rate of 50% 3 times a week.
ARS of CGF primary cells was performed at 21 days to detect osteoblast calcification. ARS was quantitated spectrophotometrically by adding 10% cetylpyridinium chloride [29 (link)]. Absorbance was measured at 562 nm by a spectrophotometer (Beckman Coulter DU800, Brea, CA, USA).
Data are represented as mean ± SD of triple measurements from three independent experiments.