Cetylpyridinium chloride monohydrate

Alkaline phosphatase (ALP) staining was performed at day 5 in BMSCs during osteogenic differentiation. Cells rinsed with phosphate-buffered saline (PBS) were fixed with 4% paraformaldehyde, and then ALP staining was performed using an ALP substrate mixture (ALP staining kit, Sigma) for 30 min in darkness. ALP activity was colorimetrically determined at 405 nm using a Sigma kit with p-nitrophenyl phosphate (pNPP) as the substrate. The protein contents were measured using a protein assay kit (Bio-Rad) based on the bicinchoninic acid (BCA) method, and the overall enzyme activity was presented as units/mg protein.
Alizarin red S (ARS) staining was performed at day 10 during osteogenic differentiation. Cells were fixed in ice-cold 70% ethanol and then stained with 3% alizarin red S solution (Sigma) for 30 min. The bound dye was then colorimetrically detected at 595 nm after de-staining with 10% cetylpyridinium chloride monohydrate (Sigma) for 20 min. The quantification of ARS staining was conducted by drawing an Alizarin Red standard curve.

Cells were seeded into 24-well plates at a concentration of 5 × 10⁴ cells/well. After 24 h, the media were replaced with 25% of the different extracted osteogenic mediums and cultured for 14 days. Alkaline phosphatase staining (ALP) was performed at day 10, and Von Kossa and alizarin red s staining were performed at day 14 [11 (link), 14 (link)]. For ALP staining, the cells were fixed and stained with TRACP & ALP double-stain kit (Takara Bio USA Inc., CA, USA). For alizarin red s staining, the cells were fixed with cold methanol and washed with deionized water. Alizarin red s solution (1% w/v) was incubated with the samples for 3 min, removed, and washed with DI water 3 times. The staining was solubilized with 10% cetylpyridinium chloride monohydrate (Sigma-Aldrich, MO, USA) solution and the absorbance was measured at 570 nm using a spectrophotometer. For Von Kossa staining, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, MO, USA) in PBS for 10 min. After rinsing with deionized water, 5% silver nitrate (Sigma-Aldrich, MO, USA) was added and the samples were exposed to UV light for 60 min. The cells were washed and rinsed with 5% sodium thiosulfate (JT Baker) 3 times before counterstaining with methyl-green (Takara Bio USA Inc., CA, USA).

TCs+, TCs- and clones at P4 were seeded at 10⁴ cells/cm² and differentiated for 14 days in osteogenic medium consisting of CM supplemented with 10 nM dexamethasone, 10 mM glycerol-2-phosphate, 150 μM L-ascorbic acid-2-phosphate and 10 nM cholecalciferol (all from Sigma- Aldrich) (de Girolamo et al. 2009 (link)). The extracellular calcified matrix deposition was measured using Alizarin Red-S staining. Cells were fixed with ice-cold 70% ethanol for 1 h and stained with 40 mM Alizarin Red S (pH 4.1; Fluka-Sigma Aldrich, MO, USA) for 15 min. The dye was extracted with 10% cetylpyridinium chloride monohydrate (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.0) and the absorbance was read at 550 nm (Perkin Elmer Victor X3 microplate reader).

Mineral deposition in BMSCs was observed by ARS after OI. Cells were treated with 4% paraformaldehyde at room temperature for 15 min, washed with PBS three times, and stained with alizarin red (10 μL/mL, Sigma) at room temperature for 30 min. The images of stained cells were captured by a microscope. For ARS quantification, cells were incubated with 10% cetylpyridinium chloride monohydrate (Sigma) for 30 min, gently shaken, and rinsed with PBS. The solution was collected and the absorbance at a wavelength of 562 nM was determined using a microplate reader.

After 14 days of osteogenic induction or 12 days of adipogenic induction, MSCs were fixed with 4% paraformaldehyde for staining. The alizarin red S (ARS) dye was made up of 1% ARS (pH 4.3) and the oil red O (ORO) dye was made up of 0.3% ORO dissoleved in 60% isopropyl alcohol. A 20‐min staining step was conducted at room temperature. Thereafter, the dye was removed, and nonspecific staining was washed away with PBS. Then, the stained cells were observed and photographed under a microscope. 10% cetylpyridinium chloride monohydrate (Sigma) was used to extract combined ARS and isopropyl alcohol was used to extract combined ORO. Then, a 200‐μL aliquot was transferred to a 96‐well plate for measurement of absorbance at 562 nm for ARS and 520 nm for ORO.