BMSCs were seeded on 24‑well plates at a density of 2.5×104 cells/well. After 80% confluence of cells, wells were randomly divided into different groups. After 21 days of osteogenic induction, BMSCs were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, and incubated with Alizarin Red S (ARS) solution (Beyotime) for 10 min. The ARS staining was extracted with 10% (w/v) cetylpyridinium chloride, and the OD value was measured at 570 nm.
Alizarin red s solution
Manufactured by Beyotime
Sourced in China
Alizarin Red S solution is a laboratory reagent used for the detection and identification of calcium in various samples. It is a bright red dye that forms a red-colored complex when it reacts with calcium ions. The solution is commonly used in histology, mineralogy, and other fields to visualize the presence and distribution of calcium in specimens.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Agilent Technologies (2 mentions)
Alp staining kit, by Beyotime (1 mentions)
Alkaline phosphatase alp, by Beyotime (1 mentions)
Bcip nbt solution, by Beyotime (1 mentions)
P nitrophenyl phosphate pnpp, by Beyotime (1 mentions)
Fsx100, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Elx808, by Agilent Technologies (1 mentions)
Alp kit, by Nanjing Jiancheng (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
11 protocols using alizarin red s solution
1
Osteogenic Differentiation of BMSCs
2
Osteogenic Differentiation Kinetics
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When BMSC confluency reached 80%, the medium was replaced with osteogenic medium. Varying concentrations of ICA were added. Total cellular proteins were extracted, and the supernatant liquid was collected for downstream assays. ALP activity was measured with an ALP Staining Kit (Beyotime) after 1 wk. Calcium mineralization was detected after 3 wk via alizarin red S (ARS) solution (Beyotime).
3
Investigating Nicotine's Impact on Osteoblasts
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To examine the effects of NIC on osteoblast differentiation and function, primary mouse BMSCs were seeded into 48-well plates at a density of 8 × 104 cells/well where they grew to 90% confluence. BMSCs were stimulated with osteogenic media containing 10 mM β-glycerophosphate, 50 μg/ml ascorbic acid, and 10−7 mM dexamethasone without or with various concentrations of NIC (12.5, 25, or 50 μM). Half of the osteogenic medium was replenished every 2 days. BMSCs were stimulated under osteogenic conditions for 7 days and the cells fixed in 4% PFA before stained for alkaline phosphatase (ALP) (Beyotime Biotechnology) activity to determine the effects of NIC on osteoblast differentiation. On the other hand, BMSCs were cultured under osteogenic conditions for 21 days and the cells fixed in 4% PFA were washed three times with 70% ethanol and stained with 1% Alizarin Red S (ARS) solution for 30 min (Beyotime Biotechnology) to determine the effects of NIC on osteoblast mineralization function. ImageJ software (NIH) was adopted to evaluate the ALP and mineralization activity of osteoblast.
4
Osteogenic Differentiation of Mouse Stem Cells
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mSSCs were trypsinized and replated in a 12-well plate at a concentration of 5 × 104 cells per well. After being incubated in complete medium for 2 days, the medium was replaced by osteogenic induction medium (Oricell, Shanghai, China). The induction medium was changed every 3 days. The alkaline phosphatase (ALP) assay and alizarin red S (ARS) staining were conducted, respectively, on 14 days and 21 days of osteogenic induction. After being fixed for 30 min at room temperature with 10% neutral-buffered formalin, mSSCs were incubated with a BCIP/NBT solution (Beyotime, Shanghai, China) or alizarin red S solution (Beyotime) at room temperature for 10 min. ALP activities were assessed using p-nitrophenyl phosphate (pNPP, Beyotime), and the OD values were measured at 405 nm. ARS staining were quantified using 10% cetylpyridinium chloride (CPC, Beyotime) in 10 mM sodium phosphate for 15 min at room temperature, and the OD values were measured at 562 nm.
5
Osteoblast Mineralization Quantification
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At the end of the calcium deposition period (21 days), OBs in the 6-well plate were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature and washed three times with PBS. Then samples were stained with 2% Alizarin Red S solution (C0138, Beyotime, Chian) at 37℃ for 30 min and washed three times with double-distilled water. Plates were air-dried before imaging under light microscopy. Mineralization intensity was measured using ImageJ software.
6
Alizarin Red S Staining of Tissues
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The spheroidal sections were fixed in 4% PFA at room temperature for 10 min. Rinse the section with deionized distilled water three times for 5 min each. Sections were incubated in 2% Alizarin red S solution (pH4.2) (Beyotime) at room temperature for 30 min. The section was rinsed with several changes of deionized distilled water for 5 min and then photographed by a microscope (Olympus FSX100).
7
Quantifying Osteoblast Mineralization
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Primary osteoblasts were stained with 2% Alizarin Red S solution (Beyotime) for 30 min. The stained images were captured using the microscope. For the quantitative assessment of the degree of mineralization, the red stain was eluted by 10% acetic acid for 30 min, and then quantified via spectrophotometric absorbance measurements of the optical density at 590 nm.
8
Osteogenic Differentiation of HBMSCs
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HBMSCs were passaged in 24-well plates and cultured in an osteogenic induction medium for 14 d. Cells were fixed in 4% paraformaldehyde (Beyotime, Shanghai, China) for 20–30 min at room temperature and then rinsed three times with PBS. Alizarin Red S solution (Beyotime) was then added and incubated at room temperature for 15 min followed by rinsing with PBS three times. ARS quantification was performed as previously described [45 (link)]. The stain was incubated with 10% cetylpyridinium chloride for 1 h at room temperature, and the solutions were collected and plated in a 96-well plate and read at 560 nm with a microplate reader (ELX808; BioTek). The results were normalised to those of the control group.
9
Osteogenic Differentiation of hDPSCs
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The hDPSCs were digested with 0.25% Trypsin-0.04% EDTA, and seeded into 24 well plates with 0.1% gelatin. In each well, 1 mL complete medium (HUXDP-90011, OriCell) was added. When the degree of cell fusion reached 60%-70%, removed the complete medium, and added 2 mL complete medium for osteogenic differentiation (HUXDP-90021,OriCell). The complete medium for osteogenic differentiation was changed every 3 days. After 21 days, removed the medium and washed with PBS. In each well, 4% neutral formaldehyde was added for 30 min. After washing with PBS, 2% alizarin red S solution (C0138, Beyotime, China) was added for 3-5 min. After washing, the cells were observed under a light microscope.
10
Osteogenic Potential of FAC and E2
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OBs were treated with FAC and E 2 for 3 days and were then stained using alkaline phosphatase (ALP) following the manufacturer's instructions (Beyotime, Shanghai, China). For alizarin red staining, cells were treated according to the experimental design. After 14 days, Cells were fixed and added with Alizarin Red-S solution (Beyotime) for 30 min. Then mineralized nodules were photographed. Cultured cells were seeded in 12-well plates and treated with FAC and E 2 . After 10 days, cells were lysed with cell lysis buffer and centrifuged at 250 g for 5 min. Aliquots of supernatant were collected to measure ALP activity and protein concentration by using an ALP kit (Jiancheng, Nanjing, China) and a BCA protein assay kit (Beyotime) respectively. The OD was measured at a wavelength of 520 nm by using the BioTek microplate reader.
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