Bcip nbt alkaline phosphatase chromogenic kit

Manufactured by Beyotime

Sourced in China

The BCIP/NBT alkaline phosphatase chromogenic kit is a laboratory reagent used for the detection and visualization of alkaline phosphatase activity in various applications, such as immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assays (ELISA). The kit provides a chromogenic substrate, consisting of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT), which produces a dark purple-blue precipitate upon the catalytic activity of alkaline phosphatase.

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7 protocols using bcip nbt alkaline phosphatase chromogenic kit

Osteogenic Differentiation of Human Bone Marrow MSCs

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BMSCs, third
to fourth generation, were seeded into 24-well plates pre-coated with
0.1% gelatin. After treatment, when cell fusion reached 60–70%,
the human bone marrow MSC osteogenic differentiation induction medium
was changed every 3 days (Cyagen, HUXMA-90021, Guangzhou, China),
and osteogenic differentiation induction was started. ARS was performed
21 days later. When obvious calcium nodules were observed in the control
group, the cell medium was removed. The cells were washed three times
with PBS, fixed with 4% paraformaldehyde, washed three times with
PBS, and then stained with ARS solution for 3–5 min. For ALP
staining, after 7 days, alkaline phosphatase dye was pre-configured
in advance according to the manufacturer’s instructions by
using the BCIP/NBT alkaline phosphatase chromogenic kit (Beyotime,
C3206, Shanghai, China). The cells were first fixed in 4% paraformaldehyde,
washed three times in PBS, and incubated for 30 min with the dye protected
from light. The cells were washed three times with PBS and placed
under a microscope and a camera for photography.

Yu C., Chen L., Zhou W., Hu L., Xie X., Lin Z., Panayi A.C., Zhan X., Tao R., Mi B, & Liu G. (2022). Injectable Bacteria-Sensitive Hydrogel Promotes Repair of Infected Fractures via Sustained Release of miRNA Antagonist. ACS Applied Materials & Interfaces, 14(30), 34427-34442.

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Biomaterial Synthesis for Bone Regeneration

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Ethanol and sodium alginate were purchased from Aladdin Reagents (China). Gluconolactone was purchased from Chembee (China). Bortezomib was purchased from D& (China). Nano-hydroxyapatite was purchased from RHAWN (China). Trizol, Radio immunoprecipitation assay (RIPA) lysis buffer, BCIP/NBT alkaline phosphatase chromogenic Kit, Alizarin Red S Staining Kit, Calcium Colorimetric Assay Kit, and Dexamethasone (≥ 99%, Reagent grade) were purchased from Beyotime Biotechnology (China). Sodium β-glycerophosphate was purchased from Macklin (China). l-ascorbic acid was purchased from Sigma (China). Calcein AM & propidium iodide (PI) probes were purchased from Life Technologies (China). Reverse Transcription Kit, SYBR Green Detection System Kit, and ECL Chemiluminescent Substrate Kit were purchased from Servicebio (China). PVDF was purchased from Millipore (USA).

Chen J., Wen J., Fu Y., Li X., Huang J., Guan X, & Zhou Y. (2023). A bifunctional bortezomib-loaded porous nano-hydroxyapatite/alginate scaffold for simultaneous tumor inhibition and bone regeneration. Journal of Nanobiotechnology, 21, 174.

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Quantitative Analysis of Early and Late Mineralization

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The early mineralization was determined based on the instructions of BCIP/NBT alkaline phosphatase chromogenic kit (Beyotime, Shanghai, China). Cells were fixed in 4% paraformaldehyde (Beyotime) for 10 min, washed three times with PBS, and then incubated with BCIP/NBT staining solution for 30 min at 37 °C in the dark. After that, the treated cells were washed twice with distilled water and photographed. Then, ALP concentration was quantitatively analyzed. Late mineralization was measured according to the instructions of the Osteoblast Mineralized Nodule Staining Kit (Beyotime). The cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS for another three times, and stained with alizarin red for 10 min. Subsequently, the treated cells were washed with distilled water to be observed and photographed microscopically, and the calcium nodules level was then quantitatively analyzed.

Jiang J., Zhang N., Song H., Yang Y., Li J, & Hu X. (2023). Oridonin alleviates the inhibitory effect of lipopolysaccharide on the proliferation and osteogenic potential of periodontal ligament stem cells by inhibiting endoplasmic reticulum stress and NF-κB/NLRP3 inflammasome signaling. BMC Oral Health, 23, 137.

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Osteogenic Differentiation of hDPSCs

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The hDPSCs were cultured with osteogenic induction medium extracts of DRCs for 14 days, and a blank control was set. For ALP staining, the cells were fixed with 4% paraformaldehyde for 30 min. Subsequently, the fixative was removed and the cells were stained using BCIP/NBT Alkaline Phosphatase Chromogenic Kit (Beyotime, China). The staining images were observed by an inverted microscope (Nikon, Japan). For the ALP activity assay, after lysis with 1% Triton X-100 (Beyotime, China) for 30 min, the ALP protein of the hDPSCs was measured by an Alkaline Phosphatase Assay Kit (Beyotime, China) and recorded as the OD value at 405 nm. Next, the total protein concentration in each sample was determined using a BCA protein assay kit (Thermo, USA) for normalization.

Zhang S., Wang X., Yin S., Wang J., Chen H, & Jiang X. (2024). Urchin-like multiscale structured fluorinated hydroxyapatite as versatile filler for caries restoration dental resin composites. Bioactive Materials, 35, 477-494.

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Osteogenic Differentiation Assay with VK2

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Cells were seeded in 6-well plates in complete medium. After the cells reached 80~90% confluence, the medium was replaced by the osteogenic medium with different concentrations of VK₂ for 7 days. Then, the cells were fixed for 20 min with 4% paraformaldehyde, and ALP staining was performed using the BCIP/NBT alkaline phosphatase chromogenic kit following the manufacturer’s instructions (Beyotime Biotechnology, Beijing, China). Cells were observed under a light microscope (Nikon, Tokyo, Japan) after staining by neutral red staining solution (Beyotime Biotechnology) for 5 min.
After treatment with VK₂ for 7 days, the cells were washed with PBS and lysed with RIPA lysis buffer (Beyotime Biotechnology). Cell lysates were analyzed for protein concentration using the BCA protein concentration assay kit (Beyotime Biotechnology). Then, ALP activity was measured using an ALP assay kit according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Wang H., Li L., Zhang N, & Ma Y. (2022). Vitamin K2 Improves Osteogenic Differentiation by Inhibiting STAT1 via the Bcl-6 and IL-6/JAK in C3H10 T1/2 Clone 8 Cells. Nutrients, 14(14), 2934.

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Osteogenic Differentiation Assay for DPCs

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To detect osteogenic differentiation ability, DPCs were cultured in osteogenic medium (OM, α-MEM supplemented with 10% FBS, 5 mM l-glycerophosphate, 100 nM dexamethasone, and 50 μg/ml ascorbic acid) in 12-well plates. After 10 days induction, the cells were fixed using 4% paraformaldehyde for 30 min and calcium deposition were stained by 2% ARS. ALP was detected at day4 after induction by using BCIP/NBT Alkaline Phosphatase Chromogenic Kit (Beyotime, China). ALP activity was measured by an ALP activity kit (Jiancheng, China) and the absorbance of each well was determined by measurements at 520 nm.

Zhou T., Chen G., Xu Y., Zhang S., Tang H., Qiu T, & Guo W. (2023). CDC42-mediated Wnt signaling facilitates odontogenic differentiation of DPCs during tooth root elongation. Stem Cell Research & Therapy, 14, 255.

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Alkaline Phosphatase Staining in Breast Cancer Cells

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Alkaline phosphatase (AP) staining was performed using the BCIP/NBT alkaline phosphatase chromogenic kit (Beyotime, Shanghai, China). Brie y, the MT-F7 and MC-F7 cells were rst washed twice with prechilled DPBS at 4°C, and then xed with 4% paraformaldehyde (P1110, Beijing Solarbio Science & Technology Co.,Ltd., Beijing, China) for 15 min at 21-26°C. The cells were then incubated with the staining solution for 30 minutes in the dark and at room temperature. Then, after rinsing twice with DPBS, the cells were photographed using an inverted light microscope (Nikon).

Xu W., Hao R., Wang J., Gao L., Han X., Li C., Zhang H., & Li X. (2022). Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells.

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