A cell suspension of 1 mL was seeded on each sample at a density of 1×104 cells/mL. After culturing for 7 and 14 days in the absence and presence of osteogenic supplements, the cells were washed and fixed. ALP staining was conducted with an ALP color development kit (Beyotime Institute of Biotechnology), and ALP activity was determined by an ALP activity detection kit (Jiancheng Bioengineering Institute, Nanjing, People’s Republic of China), according to the manufacturer’s suggested protocols.
Alp color development kit
Manufactured by Beyotime
Sourced in China
The ALP color development kit is a laboratory equipment designed for the detection and quantification of alkaline phosphatase (ALP) activity. It provides a simple and reliable method for measuring ALP levels in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alp assay kit, by Beyotime (3 mentions)
Alp activity detection kit, by Nanjing Jiancheng (2 mentions)
Alizarin red staining kit, by Beyotime (2 mentions)
Elx808, by Agilent Technologies (1 mentions)
Alp activity assay, by Beyotime (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd86, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Alizarin red solution, by Cyagen (1 mentions)
Alp activity kit, by Nanjing Jiancheng (1 mentions)
Alkaline phosphatase assay kit, by Beyotime (1 mentions)
Inverted microscope, by Zeiss (1 mentions)
Bx51trf, by Olympus (1 mentions)
21 protocols using alp color development kit
1
ALP Staining and Activity Evaluation
2
Odontoblastic Differentiation and Mineralization Assay
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The hDPSC/wt, hDPSC/control or hDPSC/shcrab cells were incubated in odontoblastic induction medium for 14 days. Mineralized nodules were assessed following alizarin red staining. Briefly, the cells in six-well plates were cultured in 1% alizarin red (pH 4.3) for 30 min at 37˚C following fixation in 4% paraformaldehyde. For ALP staining, the plates were harvested on day 7. The ALP color development kit (Beyotime Institute of Biotechnology, China) was used according to the manufacturer's protocol. All the cells were washed thrice with distilled water and then observed under a phase-contrast microscope.
3
BMSC Osteogenic Differentiation Assay
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BMSCs were seeded in 24-well plate at a density of 2 × 104 cell per well, and cultured with the extracts of fabricated sponges. After culturing for 7 days, the cells were fixed in 4% paraformaldehyde and stained using an ALP Color Development Kit (Beyotime). Moreover, a quantitative analysis about the ALP activity of BMSCs treated with extracts for 7 days was performed according to the manufacturer's instruction of ALP Assay Kit (Beyotime).
4
Quantifying Alkaline Phosphatase in MSCs
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MSCs were seeded in a 24-well plate with a density of 30%–40%, and stimulated with adenovirus respectively. For ALP staining, cells were fixed with 4% paraformaldehyde for 30 min, then, washed twice with PBS and stained with ALP Color Development Kit (Beyotime, China). Thirty minutes later, cells were observed under a bright field microscopy. As for quantitative analysis of ALP activity, cells in each treatment group were lysed by lysis buffer, then 5 μL lysis buffer, 5 μL substrate (BD Clontech), and 15 μL Lupo Buffer were mixed and incubated in light-proof condition for 20 min followed with measuring chemiluminescence signals. ALP activity was normalized by total protein concentrations in each treatment group. Each assay condition was performed in triplicate, and the results were repeated in at least 3 independent experiments.
5
Osteogenic Differentiation Assay
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Cells were passaged to 12-well plates and cultured with osteogenic induction medium when cells reached at 70% con uence. Osteogenic induction medium cultured for 5 days, cells were washed by PBS 3 times, and were xed in 4% paraformaldehyde for 20min-30min at RT. Washed by PBS 3 times, cells were stained by the ALP Color Development Kit (Beyotime). Finally, the ALP activity quanti cation was performed as previous described.
6
Alkaline Phosphatase Activity in Ameloblastic Cells
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The wild-type, control and dlk1-oe groups were seeded 5×105 cells/per well in 6-well plates in triplicate and cultured in ameloblastic induction medium for 7 days. The induction medium was replaced every 3 days. An ALP color development kit (Beyotime Institute of Biotechnology, Haimen, China) was used, according to the manufacturer's protocol. The plates were treated with an ALP staining kit for 30 min at 37°C following fixation in 4% paraformaldehyde for 15 min at 37°C. The cells were washed three times with distilled water and observed by phase-contrast microscopy (magnification ×200).
7
Osteogenic Differentiation Assay
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Cells were passaged to 12-well plates and cultured with osteogenic induction medium when cells reached at 70% confluence. Osteogenic induction medium cultured for 5 days, cells were washed by PBS 3 times, and were fixed in 4% paraformaldehyde for 20–30 min at RT. Washed by PBS 3 times, cells were stained by the ALP Color Development Kit (Beyotime). To measure ALP quantified, ALP activity was determined by ALP Activity Assay (Beyotime). Stain was lysed with lysis buffer consisted of 20 mM Tris–HCl (pH 7.5), 1% Triton X-100 and 150 mM NaCl and the conversion color of p-nitrophenyl phosphate was measured at 405/650 nm by a microplate reader (ELX808; BioTek).
8
Osteogenic Differentiation of BMSCs with Leonurine
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BMSCs at a density of 2 × 104 cells/well were seeded in 24-well plates. After 24 h, the baseline medium was replaced with osteogenic induction medium containing various concentrations of leonurine (0, 2, 5, and 10 μM). The cells were cultured in osteogenic induction medium for either 6 days (ALP staining) or 14 days (Alizarin red staining).
ALP staining was conducted to ascertain the effect of leonurine on BMSC differentiation. In brief, BMSCs were harvested after 6 days of culture and fixed with 4% paraformaldehyde for 10 min. An ALP color development kit (Beyotime, Shanghai, China) was used in the study according to the manufacturer’s protocols. Briefly, the cells were stained for 15 min and washed three times with PBS. The stained cells were subsequently observed under phase-contrast microscopy, with representative images captured.
Alizarin red staining was further performed to determine the degree of calcium deposition in BMSCs between the leonurine treatment groups at various concentrations. Briefly, BMSCs were harvested after 14 days of culture in osteogenic medium as outlined above. After fixation in 4% paraformaldehyde for 10 min, the cells were stained with Alizarin red staining kits (Beyotime, Shanghai) for 60 min and washed three times with ddH2O.
The stained cells were subsequently observed under phase-contrast microscopy, with representative images captured.
ALP staining was conducted to ascertain the effect of leonurine on BMSC differentiation. In brief, BMSCs were harvested after 6 days of culture and fixed with 4% paraformaldehyde for 10 min. An ALP color development kit (Beyotime, Shanghai, China) was used in the study according to the manufacturer’s protocols. Briefly, the cells were stained for 15 min and washed three times with PBS. The stained cells were subsequently observed under phase-contrast microscopy, with representative images captured.
Alizarin red staining was further performed to determine the degree of calcium deposition in BMSCs between the leonurine treatment groups at various concentrations. Briefly, BMSCs were harvested after 14 days of culture in osteogenic medium as outlined above. After fixation in 4% paraformaldehyde for 10 min, the cells were stained with Alizarin red staining kits (Beyotime, Shanghai) for 60 min and washed three times with ddH2O.
The stained cells were subsequently observed under phase-contrast microscopy, with representative images captured.
9
Osteogenic Differentiation Assay of hPDLSCs and hUCMSCs
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After 7 days of osteogenic induction, ALP staining of hPDLSCs-CA and hUCMSC-CA was performed by an ALP color development kit (Beyotime, Shanghai, China) and ALP activity was quantified by an ALP activity detection kit (Jiancheng Bioengineering, Nanjing, China). Quantitative real-time PCR (RT-qPCR) was performed to examine the mRNA expression of ALP, Runx2 and OCN as well as extracellular matrix-related gene, fibronectin, integrin-β and collagen type I. In addition, BSP and OPN, which are strongly expressed in the native periodontium in bone and cementum, especially acellular cementum, were also detected by the RT-qPCR. Briefly, total cellular RNA was isolated from hPDLSCs-CA and hUCMSCs-CA using Trizol Reagent (Invitrogen) according to the manufacturer's standard instructions. Reverse transcription of mRNA and PCR reaction were performed as described previously 40 (link). Primer sequences used in this experiment are listed in Table 1 . Also, expression of BSP, OPN, fibronectin, collagen type I, OCN and osterix were detected by immunofluorescence in both cell-aggregates during the induction period. The experimental process and analysis method were as previously described 32 (link). The experiment was repeated at least three times.
10
Polymer-Based Tissue Engineering Scaffold
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PP was provided by the Kangmei Pharmaceutical Co. Ltd. (Guangdong, China).PEGDA (containing 400–600 ppm MEHQ stabilizer) was purchased from the Shanghai Yuanye Biotechnology Co. Ltd. (China). CMC was purchased from the Zhejiang Aoxing Biotechnology Co. Ltd. (China). GO was obtained from the Xianfeng Nanomaterials Technology Co. Ltd. (Jiangsu, China). 2-Hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Photoinitiator 2959) was purchased from Aladdin Reagent (Shanghai, China). Interleukin (IL)-6, IL-4, IL-10, transforming growth factor-β (TGF-β), and tumor necrosis factor-α (TNF-α) ELISA kits were purchased from YIFEIXUE BIOTECH (Nanjing, China). FITC-conjugated anti-CD86, PE-conjugated anti-CD206, recombinant anti-iNOS, anti-osteocalcin (OCN), and anti-alkaline phosphatase (ALP) antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CD68 rabbit polyclonal, bone morphogenetic protein-2 (BMP-2) polyclonal, runt-related transcription factor 2 (RUNX2), and osteopontin (OPN) polyclonal antibodies were purchased from AiFang Biological (Hunan, China). Osteogenic inducting fluid was purchased from Procell (Wuhan, China). ALP Color Development Kit and Alizarin red S (ARS) kit were purchased from Beyotime (Shanghai China).
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