The nHA (purity >97%, CAS 1306-06-5) was purchased from Aladdin Industrial Inc. (Shanghai, China). Cell Counting Kit-8 (CCK-8), Annexin V-FITC/PI apoptosis detection kit, bicinchoninic acid (BCA) protein assay kit, Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum (FBS), penicillin and streptomycin were supplied by Meilunbio (Dalian, China). EasySep™ mouse T cell isolation kit was obtained from StemCell Technologies (Vancouver, CA, United States). Anti-mouse CD3 (Clone 17A2), anti-mouse CD28 (Clone 37.51) were purchased from Peprotech (Westlake Village, CA, United States). Anti-mouse CD4, phycoerythrin (PE, Clone GK1.5), Anti-mouse CD8α, phycoerythrin-cyanine 7 (PE-Cy7, Clone 53-6.7) and flow cytometry staining buffer was purchased from Multi Sciences Biotech, Co., Ltd. (Hangzhou, China). 5-(and-6)-Carboxyfluorescein diacetate, succinimidyl ester (CFSE) was obtained from Abbkine Bioadvisers. Alizarin red S (ARS) staining kit for osteogenesis was purchased from Beyotime (Shanghai, China). Dimethyl sulfoxide (DMSO) and RPMI 1640 culture medium were obtained from Solarbio (Beijing, China). Deionized (DI) water was used in all experiments. All chemicals were used without additional purification.
Alizarin red s staining kit for osteogenesis
Manufactured by Beyotime
Sourced in China
The Alizarin Red S Staining Kit for Osteogenesis is a laboratory product designed to detect and quantify the mineralization of cells during the process of osteogenesis, or bone formation. The kit contains the Alizarin Red S dye, which binds to calcium deposits in the extracellular matrix, allowing for the visualization and analysis of the degree of mineralization in cell cultures.
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Lab products found in correlation
Bcip nbt alkaline phosphatase color development kit, by Beyotime (2 mentions)
Bicinchoninic acid bca protein assay kit, by Meilun (1 mentions)
Dulbecco s modified eagle s medium dmem, by Meilun (1 mentions)
Rpmi 1640 culture medium, by Solarbio (1 mentions)
Easysep mouse t cell isolation kit, by STEMCELL (1 mentions)
Fetal bovine serum (fbs), by Meilun (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Meilun (1 mentions)
Cell counting kit 8 cck 8, by Meilun (1 mentions)
Penicillin, by Meilun (1 mentions)
Streptomycin, by Meilun (1 mentions)
Trypsin, by Meilun (1 mentions)
Flow cytometry staining buffer, by MultiSciences Biotech (1 mentions)
Quantifluor dsdna system kit, by Promega (1 mentions)
P nitrophenyl phosphate, by Merck Group (1 mentions)
Ckx53, by Olympus (1 mentions)
Alkaline phosphatase assay kit, by Beyotime (1 mentions)
Mdi6000b, by Leica camera (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Bcip nbt alp colour development kit, by Beyotime (1 mentions)
5 protocols using alizarin red s staining kit for osteogenesis
1
Nanohydroxyapatite Cytotoxicity and Immunomodulation
2
Osteogenic Differentiation of Cells
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Cells were cultured for 7 days in differentiation medium, and the light treatment was repeated once a day for 7 days. ALP activity was measured using the p-nitrophenyl phosphate (Sigma, U.S.A.) method as previously described.95 The ALP activity was normalized by DNA content per sample using a QuantiFluor dsDNA System kit (E2670, Promega Corporation, Madison, U.S.A.). For ALP staining, cells were fixed with 4% paraformaldehyde for 30 min and stained with the BCIP/NBT alkaline phosphatase color development kit (C3206, Beyotime, China). For mineralization staining, cells were cultured for 14 days in differentiation medium with or without light irradiation, followed by staining with the Alizarin red S (ARS) staining kit for osteogenesis (C0148S, Beyotime, China). Images were taken using an optical microscope (Nikon, Japan).
3
Osteogenic Potential of Zinc-Doped Chitosan Hydrogels
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The cells (104 cells/mL) were cultured with HA, CHA-0, CHA-L, CHA-M, CHA-H, and CHA-G in 12-well plate. After 14 days of culture, the alkaline phosphatase assay kit (Beyotime, China) was used to measure alkaline phosphatase (ALP) activity, and the optical density (OD) value was measured with a microplate reader at 405 nm. Cellular samples were stained with BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China), after being fixed with 4% paraformaldehyde for 20 min.
After 21 days of incubation, the samples were stained using the Alizarin Red S Staining Kit for Osteogenesis (Beyotime, China) for 30 min, followed by a rinse with distilled water. Then, an inverted optical microscope (CKX53, Olympus, Japan) was used to observe the stained cells. Afterwards, the 10% (w/v) cetylpyridinium chloride (diluted with PBS; Aladdin, China) was used to quantify the Alizarin red staining (ARS). The absorbance was measured at 562 nm. In addition, the amount of Zn2+ released from Zn-doped samples in the collected medium was detected using commercial kits (Zinc, China).
After 21 days of incubation, the samples were stained using the Alizarin Red S Staining Kit for Osteogenesis (Beyotime, China) for 30 min, followed by a rinse with distilled water. Then, an inverted optical microscope (CKX53, Olympus, Japan) was used to observe the stained cells. Afterwards, the 10% (w/v) cetylpyridinium chloride (diluted with PBS; Aladdin, China) was used to quantify the Alizarin red staining (ARS). The absorbance was measured at 562 nm. In addition, the amount of Zn2+ released from Zn-doped samples in the collected medium was detected using commercial kits (Zinc, China).
4
Quantifying Osteogenic Differentiation
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Cells on scaffolds were examined after 14 and 21 days of culture using the Alizarin Red S Staining Kit for Osteogenesis (#C0138, Beyotime Biotechnology, Shanghai). Briefly, cells on scaffolds were fixed with 4% paraformaldehyde for 20 min, washed 3 times with PBS, stained with Alizarin Red S staining solution at room temperature for 30 min, washed with deionized water and photographed for mineralized nodules. After that, 10% cetylpyridinium chloride was added to lyse the mineralized nodules and the absorbance was measured at 620 nm for semi-quantitative analysis.
5
Osteogenic Differentiation in 2D and 3D
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For investigating the extent of osteogenic differentiation in the 2D and 3D environment, BMSCs with and without hydrogel were cultured in osteogenic medium without exosomes for a fixed time interval. To measure ALP expression, cells were fixed with 10% formalin, stained using BCIP/NBT ALP Colour Development Kit (Beyotime), and imaged with an inverted fluorescence microscope (DMI6000 B, Leica, Germany). To quantitate ALP activity, cells were tested with an ALP Assay Kit (Beyotime). Absorbance was measured at 405 nm. To investigate mineral deposition, cells were fixed with formalin and stained with Alizarin Red S Staining Kit for Osteogenesis (Beyotime) for approximately 20 min, washed three times with dH2O and stained with ARS for 20 min at room temperature. Cells were imaged with a Leica MDI6000 B fluorescence microscope. After several washes with dH2O, the stain was desorbed with 200 µL 10% cetylpyridinium chloride (Sigma, Germany) for 1 h. The dye was collected, and the absorbance was read at 590 nm using a spectrophotometer.
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