Alizarin red stain

MSCs were seeded on a 5 μM ELP-coated 6-well plate and cultured to grow to 60% confluence. After 2 days, cells were cultured in DMEM medium containing 10% FBS supplemented with 10⁻³ M dexamethasone (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 50 μg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β- glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), and 0.1 μM vitamin D (Enzo Life Sciences, Farmingdale, NY, USA) to induce osteogenesis. Medium changes occurred every 3 days, and the success of differentiation was assessed 7, 14, and 21 days after induction. Alizarin Red stain (Rowley Biochemical Institute, Danvers, MA, USA) was used to examine the calcium deposition on the ELP-coated plates 14 days after induction. The cells were rinsed twice with PBS, fixed with 60% isopropanol (Sigma-Aldrich, St. Louis, MO, USA) for 1 min, washed three times with ddH₂O, and stained with 2% Alizarin Red stain for 5 min. Cells were then washed extensively with ddH₂O and imaged using a bright field microscope (Nikon, Tokyo, Japan).

Pre-treatment of assay microplates (Falcon^®, USA) was performed with the coating of multiple 12-well cell culture microplate wells using 50 µg/mL rat tail collagen I (Corning^®, USA). hFPTs and ASC-F cells were then seeded at a relative viable density of 1.5 × 10³ cells/cm² in the microplates in FBS- or HPL-supplemented growth medium, as described previously, respectively. Cultures were appropriately maintained in both incubation conditions (i.e., normoxia and hypoxia) until cell monolayers attained 80% confluency (i.e., 6 ± 2 days). Thereafter, the specific culture medium was replaced with a specific osteogenic induction medium, composed of high-glucose DMEM (Gibco™, USA) supplemented with 5% v/v HPL (Stemulate^®, USA), 80 µg/mL VitCp (Sigma-Aldrich^®, USA), 5 mM β-glycerophosphate (Sigma-Aldrich^®, USA), and 100 nM dexamethasone (Acros Organics™, USA). The induction medium was exchanged twice weekly for a period of 21 days for the different cell types and in both incubation conditions. At the end of the induction period, the cells were either fixed with a 4% formalin solution and stained with a classical Von Kossa staining procedure (Sigma-Aldrich^®, USA) or were fixed with 70% ethanol and stained with Alizarin Red stain (Sigma-Aldrich^®, USA) for revelation of mineralized matrix accumulation. Following staining, assay plates were photographed as described previously.

ASCs were grown to confluence in 6-well plates, and osteogenic differentiation was induced using osteogenic medium (DMEM 10%FBS, 0.1µM dexamethasone, 10 mM β-glycerol phosphate, and 50 μM Ascorbic Acid). At day 21 post-differentiation, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and stained with Alizarin Red Stain (Sigma-Aldrich, St. Louis, MO, USA). Quantification of staining was performed using Image J software.

Calcium and proteoglycan content of mixed populations of ATDC5-ctrl, ATDC5-wtTUFT1, and ATDC5-mutTUFT1 cells were determined by staining the cell layers with Alizarin red stain (Sigma, St Louis, USA) or Alcian blue stain (Sigma, St Louis, USA), respectively. Cells were grown 15 days after their mineralization state was analyzed. To evaluate calcium concentration, ATDC5 cells were fixed with 4% paraformaldehyde (PFA) for 5 min at 4°C, washed with PBS, stained with 2% Alizarin red (pH 4.2) for 5 min in room temperature, washed with distilled water and bound dye was extracted with 10% cetylpyridium chloride for 10 min. Optical density (OD) of the samples was determined at 570 nm by spectrophotometry. Proteoglycan content was analyzed by washing ATDC5 cells with PBS, fixing with 95% methanol for 20 min, staining with 1% Alcian blue 8GX (Sigma, St Louis, USA) in 0.1M HCl overnight and rinsing with distilled water. Cell cultures were extracted with 6M guanidine-HCl for 6h at room temperature and the released dye was evaluated by measuring the OD at 630 nm by spectrophotometer.

The human aortic valve leaflet was dewaxed and rinsed in 1 × PBS; sections were stained with 1% Alizarin Red stain (Sigma) for 1 h, washed with water, mounted using xylene-based mounting medium, and imaged.
Staining of calcium nodules generated by osteogenically differentiated hVICs was performed by fixing cells in PFA and staining with 1% Alizarin Red stain for 10 min, washing in water, and imaging with a microscope. To quantify the cells, they were washed with 10% acetate at room temperature for 30 min, and the absorbance was determined at 405 nm.

MC3T3-E1 osteogenic cells were cultured on recombinant CTGF (ProSpec) or 1% BSA (Fisher Scientific) coated 48-well plates (Falcon Becton Dickinson) and terminated on day 35 to evaluate osteoblast mineralization. Cells were washed with ddH₂O and fixed with 10% paraformaldehyde in phosphate buffer for 15 minutes at room temperature. After washing cells with 1X PBS, 40 mM Alizarin red stain (Sigma-Aldrich) was added to wells and incubated at room temperature for 15 minutes. Cells were washed with ddH₂O and air-dried. Images were taken using a Nikon Eclipse TE300 inverted microscope. ImageJ software was used to measure nodule area.