Cells were cultured in the osteogenic-/adipogenic-inducing medium to confirm the capability of multidirectional differentiation. The components of the inducing medium were described in our previous research [21 (link)]. After induction on day 21, 4% paraformaldehyde was used to fix cells. Then, Alizarin Red Staining kit (Sigma-Aldrich, St. Louis, USA) and Oil Red O kit (Solarbio, Beijing, China) were implemented to stain cells following the instructions of the manufacturers.
Alizarin red staining kit
Manufactured by Merck Group
Sourced in United States
The Alizarin red staining kit is a laboratory product manufactured by Merck Group. It is used for the histological staining of calcium deposits in tissue samples. The kit contains the necessary reagents and instructions for the Alizarin red staining technique.
Automatically generated - may contain errors
Lab products found in correlation
Oil red o kit, by Solarbio (1 mentions)
Alp staining kit, by Promega (1 mentions)
Alkaline phosphatase alp staining kit, by Merck Group (1 mentions)
Ars solution, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
10 protocols using alizarin red staining kit
1
Multilineage Differentiation Evaluation
2
Evaluating SGPA's Effect on MC3T3-E1 Mineralization
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The MC3T3-E1 cells were cultured in ODM in the presence of different concentrations SGPA for 21 days, then the mineralization function of OBs was evaluated with alizarin red staining kit (Sigma–Aldrich). The staining procedures were performed according to the manufacturer's protocols. Representative histological images were obtained under an inverted microscope (Nikon, Tokyo, Japan).
3
Alizarin Red Staining for Mineralization
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After 21 d of osteogenic induction, cell mineralization was detected using the alizarin red staining kit (Sigma-Aldrich). MC3T3-E1 cells were stained with 2% alizarin red (pH = 4.2) for 10 min, rinsed with distilled water, and viewed with microscopy (Olympus) to observe mineralized nodules.
4
Osteogenic Differentiation of MC3T3-E1 Cells
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MC3T3-E1 cells were seeded in 24-well plates at a density of 5 × 104. After that, we use 1 ml of conditioned medium per well (extraction of the LDH/CS scaffolds and SrFe12O19-LDH/CS scaffolds). Control, LDH/CS, and SrFe12O19-LDH/CS groups were prepared, and the cells were further cultured for 7 and 21 d. After that, the cells were fixed with 4% paraformaldehyde. Then the samples were washed three times with PBS for 10 min each time. Finally, the samples were stained using an ALP kit (Hongqiao, Shanghai, China) and an Alizarin Red staining kit (Sigma-Aldrich, Darmstadt, Germany) according to the reagent manufacturer's instructions.
5
Osteoblast Differentiation and Mineralization by EVO
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To determine the effect of EVO on the differentiation and mineralization of osteoblasts, primary osteoblasts were isolated from the calvariae of neonatal mice by digestion with 0.1% collagenase and 0.2% dispase. The use of mice was conducted in accordance with an ethic approval RA/3/100/1244 by the Animal Ethics Committee of the University of Western Australia. Cells from passages 1‐2 were used for the present study. α‐MEM media with 10% FBS, 1% antibiotic mixture of penicillin and streptomycin, 10−8 mmol/L dexamethasone (DEX), 50 μg/mL ascorbic acid (A.A) and 10 mmol/L β‐glycerophosphate (β‐Gly) were used to stimulate the differentiation of osteoblasts. Meanwhile, 5 and 10 μmol/L EVO were added to the medium during osteoblastic differentiation. Alkaline phosphatase (ALP) assay, an early differentiation marker of osteoblasts, and alizarin red staining assay were performed using an ALP staining kit (Promega, WI, USA) and alizarin red staining kit (Sigma, St. Louis, MO, USA) separately on days 7 and 21 of culture according to the manufacturers’ suggested protocols.
6
Osteoclast Differentiation Assay
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Psoralea corylifolia was purchased from Zhongda TCM Store (Shanghai, China). NBIF (purity ≥98%) was ordered from Shidande Company (Shanghai, China,http://www.nature‐standard.com/product/html/1138.html ). RAW 264.7 cells were supplied by Shanghai Institutes for Biological Sciences (Shanghai, China). Alpha‐modified minimal essential medium (α‐MEM) and foetal bovine serum (FBS) were obtained from Hyclone (Logan, UT, USA). Murine M‐CSF and RANKL were ordered from R&D Systems (MN, USA). Cell Counting Kit‐8 (CCK8), Tartrate‐Resistant Acid Phosphatase (TRAP) staining kit, Alkaline Phosphatase (ALP) staining kit and Alizarin Red staining kit were provided by Sigma‐Aldrich (St. Louis, MO, USA). C57BL/6 female mice (8‐week‐old, weight 20‐25 g) were ordered from Slaccas (Shanghai, China).
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Psoralea corylifolia was purchased from Zhongda TCM Store (Shanghai, China). NBIF (purity ≥98%) was ordered from Shidande Company (Shanghai, China,
7
Alizarin Red Staining for Osteoblast Mineralization
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Alizarin red staining kit (Sigma-Aldrich) was used to test the mineralization function of OBs on day 21. The staining procedures were done according to the manufacturer's suggested protocols. Pictures of histological representatives were taken using a Nikon optical lens (Nikon, Tokyo, Japan). To assess the Alizarin red S staining, 10% cetylpyridinium chloride was added into every well and cells were incubated for 20 min. The number of bone nodules per well was used to evaluate the effect of osteoblastogenesis.
8
Quantifying Cellular Mineralization
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The capability of mineralization of corresponding cells was assessed in 6-well plates using Alizarin red staining. The indicated cells were xed with ice-cold 70% ethanol and stained with Alizarin red staining kit (Sigma-Aldrich, MO, USA) to detect the calci cation according to the manufacturer's protocols. ImageJ 1.8.0 software was applied to detect the percentages of positive areas, and then quantify the mineralized areas.
9
Quantitative Assessment of Mineralization
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Mineralization was evaluated by quantifying the formation of calcium phosphate in cells using an Alizarin Red Staining (ARS) kit (Sigma-Aldrich; Merck KGaA). Briefly, the cells were cultured in 12-well culture plates at a density of 4×104 cells per ml. After 21 days, the cultured cells were fixed with 95% ethanol for 10 min at room temperature. To stain the calcium deposits, 2% ARS solution (Sigma-Aldrich; Merck KGaA) was applied for 15 min at room temperature. To measure the degree of mineralization, the ARS released from the cell matrix was incubated in cetyl pyridinium chloride for 15 min at room temperature and quantified by spectrophotometry at 540 nm.
10
Osteogenic Differentiation of BMMSCs
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BMMSCs at passage 2 were prepared for osteogenic induction, and the osteogenic medium was used for BMMSC culture which contained complete culture medium plus ascorbic acid (50 mg/L; Sigma-Aldrich, MO, USA), β-glycerophosphate (10 mM; Sigma-Aldrich), and dexamethasone (10 nM; Sigma-Aldrich), with fresh OM replaced every three days. After two weeks of induction, 4% paraformaldehyde (Solarbio, Beijing, China) was used for cell fixing. The formation of mineralized nodule was detected using Alizarin Red Staining (ARS) kit (Sigma-Aldrich). Quantification analysis of positively staining areas was performed by Image Pro Plus 6.0v (Media Cybernetics, CA, USA) and presented by ratio.
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