C3H10 cells were osteogenically induced in a DMEM medium comprising 50 μg/mL ascorbic acid and 10 μM β-glycerophosphate (Sigma-Aldrich). After osteogenic induction for 7 days, alkaline phosphatase (ALP) staining was performed with BCIP/NBT kit (CWBIO, Beijing, China), and the ALP activity was quantified by an alkaline phosphatase detection kit (Beyotime, Nanjing, China). After 14 days of osteogenic induction, alizarin red S (ARS) staining was performed with 1% alizarin red staining solution (Sigma-Aldrich) to evaluate the calcium deposition in the extracellular matrix. To quantify the mineralization, staining was eluted with 10% glacial acetic acid and the absorbance was measured at 405 nm by a microplate reader (Bio-Tek Instruments).
Bcip nbt kit
Manufactured by CWBIO
Sourced in China
The BCIP/NBT kit is a substrate system used for the colorimetric detection of alkaline phosphatase-conjugated detection reagents in immunoassays and other related applications. The kit provides a ready-to-use solution that produces a dark-blue insoluble precipitate upon reaction with the alkaline phosphatase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Agilent Technologies (3 mentions)
β glycerophosphate, by Merck Group (3 mentions)
Alizarin red staining solution, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Alkaline phosphatase detection kit, by Beyotime (1 mentions)
Alkaline phosphatase assay kit, by Beyotime (1 mentions)
Alizarin red s kit, by Solarbio (1 mentions)
Alizarin red s solution, by Merck Group (1 mentions)
6 protocols using bcip nbt kit
1
Osteogenic Differentiation of C3H10 Cells
2
Osteogenesis Induction and Quantification
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Osteogenesis induction of OB cells was performed in DMEM medium containing 50 μg/mL ascorbic acid and 10 μM β-glycerophosphatester (Sigma-Aldrich, St. Louis, MO, USA). After 7 days of osteogenic induction, the expression levels of bone-specific genes were detected. After 14 days of osteogenic induction, alkaline phosphatase (ALP) staining was performed with a BCIP/NBT kit (CWBIO, Beijing, China), and ALP activity was quantified with an alkaline phosphatase assay kit (Beyotime, Nanjing, China). Alizarin red S (ARS) staining was performed with 1% alizarin red staining solution (Sigma-Aldrich, Shanghai, China) 21 days after osteogenesis induction to observe the mineralization deposition in the extracellular matrix. To quantify the mineralization, the absorbance at 405 nm was measured with a microplate reader (Bio-Tek Instruments) by elution staining with 10% glacial acetic acid.
3
Osteogenic Differentiation Staining
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For ALP staining, osteoblasts were stained with a BCIP/NBT Kit (CWBIO, China) after 7 or 14 days of osteogenic induction. For Alizarin red S staining, osteoblasts were stained with an Alizarin red S Kit (Solarbio, China) after 7 or 14 days of osteogenic induction. ALP and Alizarin red S staining were performed according to the manufacturer's instructions.
4
Osteogenic Differentiation of Mouse MSCs
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Mouse mesenchymal stem cells MC3T3-E1 were cultured for a period of 7 days in T007-containing a-MEM, 50 mg / mL-1 ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) and 10 mM b-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA) as indicated or 21 days. The medium was revitalized every additional day. Following, the cells were stained with BCIP / NBT kit (CWBIO, Beijing, China) to detect ALP and 2% Alizarin Red S solution (pH 4.1) (Sigma-Aldrich, St. Louis, MO, USA) to visualize calcium deposition in the extracellular matrix. Mineralization was quantitated via measurements of the absorbance of the dye at 562 nm on the microplate reader after decolorization with ethyl pyridine chloride (Wako Pure Chemical Industries Ltd., Osaka, Japan).
5
Effects of MDHB on Osteogenic Differentiation
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Mouse MC3T3-E1 MSCs were cultured in α-MEM supplemented with 10% FBS. Cells were then cultured in osteogenic medium (1 mM β-glycerophosphate, and 5 mM L-ascorbic acid 2-phosphate) with different concentration of MDHB (0, 10, 20 μM) for 7 days. The medium was changed every other day. Then cells were stained with the BCIP/NBT kit (CWBIO, Beijing, China) to detect ALP staining (Sigma-Aldrich).
6
Osteoblast Differentiation and Mineralization
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Pre-osteoblasts were extracted from cranium of 1-week-old C57BL/6J and were cultured in DMEM containing 10% FBS and 1% penicillin and streptomycin. Mature osteoblasts were induced with 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), and the medium was changed every two days. Then, cells were stained with BCIP/NBT kit (CWBIO, Beijing, China) to detect ALP activity after 7 days. And 1% Alizarin red S (ARS) solution (pH 4.1) (Sigma-Aldrich) was applied to visualize calcium deposition in the extracellular matrix after 21 days. Mineralized calcium nodules were quantitatively assessed by measuring the stained extracts at 570 nm absorbance using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).
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