After culturing for 4, 7 and 14 days, the cells on both materials were harvested and then lysed with RIPA Lysis Buffer (Boster, Wuhan, China). The supernatants were collected and centrifuged at 8000 rpm and 4°C for 10 min. The total proteins in the specimens were measured using a PierceTM BCA Protein Assay Kit (Thermo Scientific, USA). ALP contents in the specimens were tested by a commercialized ALP assay kit (Biyuntian, China) according to the specifications. The relative ALP per unit protein in the specimens was calculated to normalize the data.
Alp assay kit
Manufactured by Beyotime
Sourced in China
The ALP assay kit is a laboratory tool used to measure the activity of the enzyme alkaline phosphatase (ALP) in biological samples. The kit provides the necessary reagents and protocols to quantify ALP levels accurately and reliably.
Automatically generated - may contain errors
Lab products found in correlation
Bcip nbt alp color development kit, by Beyotime (14 mentions)
Bca protein assay kit, by Beyotime (13 mentions)
Alp staining kit, by Beyotime (12 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (9 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Alizarin red, by Merck Group (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Bcip nbt alp staining kit, by Beyotime (3 mentions)
Alp color development kit, by Beyotime (3 mentions)
Ascorbic acid, by Merck Group (3 mentions)
Alizarin red staining, by Merck Group (3 mentions)
β glycerol phosphate, by Merck Group (3 mentions)
Bca protein assay reagent, by Beyotime (3 mentions)
Bcip nbt alkaline phosphatase alp color development kit, by Beyotime (2 mentions)
Bca protein quantitation kit, by Beyotime (2 mentions)
Nbt bcip staining kit, by Beyotime (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Cetylpyridinium chloride, by Merck Group (2 mentions)
Bcip nbt alp colour development kit, by Beyotime (2 mentions)
Triton x 100, by Merck Group (2 mentions)
150 protocols using alp assay kit
1
ALP Activity Quantification
2
Quantifying Osteogenic Potential of MC3T3-E1 Cells
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The alkaline phosphatase (ALP) activity of MC3T3-E1 cells was estimated by using the ALP assay kit (Biyuntian, China). Briefly, scaffolds were placed in 24-well plate and MC3T3-E1 cell suspension at a density of 3 × 105 cells mL−1 was seeded onto each scaffolds and then cultured with osteogenesis induction medium at 37 °C for 3, 7, and 14 days. At each time point, cells were lysed by Western and immunol precipitation (IP) without inhibitors (Biyuntian, China), protein concentration were assayed by BCA Protein Concentration Assay Kit (Boster, China). The concentration of protein of each sample was diluted to the same for the relative quantitative measurements of ALP according to manufacturer's instruction. The absorbance at 405 nm was measured with a microplate reader.
3
ALP Activity Assay of MC3T3-E1 Cells
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After 7 days of osteogenic differentiation, MC3T3-E1 cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. The cells were stained with BCIP/NBT Alkaline Phosphatase (ALP) Color Development Kit (Beyotime) for 30 min at room temperature. After washing with PBS, images were captured using an Olympus IX51 microscope (Olympus Corporation, Tokyo, Japan). ALP activity was measured using an ALP assay kit (Beyotime) and normalized to the protein concentration.
4
Whey Peptide Enhances Osteoblast Differentiation
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The MC3T3-E1 preosteoblast cells (subclone 4, CRL-2593), bought from ATCC (Manassas, VA, USA) were seeded on 6-well plates at a density of 1 × 105 cells per well and were incubated in growth medium (α–MEM medium supplemented with 10% FBS and 1% penicillin/streptomycin, Hyclone/Thermo Fisher, Waltham, MA, USA). After the cells reached ~80% confluency (about 48 h), 0, 5, 10, 20, 40, and 80 μM whey peptide TPEVDDEA were, respectively, added into the growth medium and co-cultured with cells for 72 h to induce differentiation. Then, radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, Nantong, China) was added to each well to collect cell lysates. The ALP activity, which is the differentiation marker of osteoblasts, was evaluated by an ALP assay kit (Beyotime) and was represented as the unit activity per mg of BCA protein content.
5
MXene Regulates Osteogenic Differentiation
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ALP activity assays were performed using an ALP assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The hPDLCs (5.0 × 10 4 cells/well) were seeded in 24-well plates. After culturing in a growth medium overnight, the cells were incubated with MXene at various concentrations in an osteogenic differentiation medium. On day 7, the cells were washed twice with PBS twice and lysed in the assay buffer, followed by centrifugation at 1.5 × 10 4 rpm to remove the insoluble substance. Then, 50 μl of the supernatant from each sample was added to 96-well plates and mixed with 50 μl of chromogenic agent solution. The plate was incubated at 37 • C for 20 min and protected from light. The reaction was stopped by adding 100 μl of stop solution. The absorbance was measured at 405 nm using a SpectraMax M3 microplate reader. A standard curve was constructed, and the ALP activity level was expressed as compared to the control.
ALP staining was performed on day 7. The plates were washed with PBS and then fixed with 4% paraformaldehyde for 30 min. The cells were then stained using a BCIP/NBT ALP staining kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions.
ALP staining was performed on day 7. The plates were washed with PBS and then fixed with 4% paraformaldehyde for 30 min. The cells were then stained using a BCIP/NBT ALP staining kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions.
6
ALP Expression in C3H10T1/2 Cells
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C3H10T1/2 cells (2 × 104 cells/well) were seeded in 12-well culture plates for 1 day and then the culture medium were replaced by the various extracts. After further cultured for 3 and 7 days, the expression of ALP was stained using the BCIP/NBT ALP color development kit (Beyotime, China) and the intracellular ALP activities were quantitatively assayed using ALP Assay Kit (Beyotime Biotechnology, China). Total protein was measured by BCA protein quantitation kit (Beyotime, China). The ALP activity was normalized with total protein content.
7
Adenoviral Transduction of iSCAPs
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The iSCAPs were seeded in 24-well plates and infected with indicated multiplicty of infection (MOI) values of AdGFP, AdBMP9, or AdsiBMP9. Polybrene (10 μg/mL) was used to enhance transduction efficiency for adenoviral infection. At 48 h after the infection, GFP signal or red fluorescent protein (RFP) signal of the infected iSCAPs was assessed under a fluorescence microscope (Carl Zeiss Microimaging GmbH, Gottingen, Germany). The infection efficiency was indicated by the GFP or RFP expression proportion of the cells. Alkaline phosphatase (ALP) histochemical staining was carried out by using the NBT/BCIP staining kit (Beyotime-Bio, China), and ALP activity was assessed quantitatively with the ALP assay kit (Beyotime-Bio, China) on day 5. Each assay condition was performed in triplicate, and the results were repeated in at least three independent experiments. ALP activity was normalized by total cellular protein concentrations determined by the bicinchoninic acid Protein Assay Kit (Beyotime-Bio, China).
8
Osteoblast Differentiation of BMMSCs
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BMMSCs were cultured on nHACM loaded with BMP-9 and treated with 50 μg/ml ascorbic acid, 5 mM β-glycerophosphate, and 10 nM dexamethasone to induce osteoblast differentiation. The culture medium was changed every 2 days. After induction for 1, 4, 7, and 10 days, ALP activity was determined in cell lysates by an ALP assay kit (Beyotime, Shanghai, China), and absorbance was read at 520 nm using an ELISA plate reader.
9
Alkaline Phosphatase Activity Assay
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At indicated time points, the cells were collected and washed with PBS, followed by centrifugation at 300 ×g for 7 min. The cells were lysed with 0.2% Triton X-100 lysis. After centrifugation at 300 ×g for 7 min, the supernatant was discarded. Reaction solution within the ALP assay kit (Beyotime) was added into the precipitation and incubates the cells at 37°C for 30 min. The absorbance at 450 nm was added with the Multiskan GO microplate reader (Thermo) to determine the ALP activities. Each group had 6 replicate wells, and the ALP activity standard curve was calculated and obtained accordingly.
10
Naringin Enhances Osteogenic Differentiation in Murine BMSCs and MC3T3-E1 Cells
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Mouse bone marrow mesenchymal stem cells (BMSCs) were obtained from 1-month-old BALB/c and Kunming mice purchased from Kunming Ainimo Experimental Animal Co., Ltd. (Kunming, China). The differentiation of BMSCs requires osteogenic induction solution, which comprises 10 mmol/L β-sodium glycerophosphate (Sigma), 50 mg/L vitamin C and 0.1 μmol/L dexamethasone (Meilunbio, Dalian, China). MC3T3-E1 cells were purchased from Kunming Cell Bank, Chinese Academy of Sciences. After culture in α-MEM (HyClone, Logan, UT, USA), the cells were transferred to 12-well plates, cultured for 12 hours, starved for 12 hours, and then treated with 10 μL of DMSO (10 μM), 1 μM, or 0.1 μM naringin (Sigma) and 100 ng/mL recombinant human bone morphogenetic protein-2 (rhBMP2, Peprotech, Cranbury, NJ, USA). The samples were collected at different time points. The mRNA expression of ALP, collagen I (ColI), osteocalcin (OC), OPG, osteopontin (OPN), and runt-related transcription factor 2 (RUNX2) in BMSCs and MC3T3-E1 cells were detected by qRT-PCR. ALP activity was detected with an ALP Assay Kit (P0321S; Beyotime, Shanghai, China), and the concentration of the osteogenic protein OC was detected by ELISA (CSB-E17681m; Cusabio, Wuhan, China).
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