The cholestatic liver tissue whole lysate containing protease and phosphatase inhibitors or only containing protease inhibitor were treated with or without alkaline phosphatase (10μg total protein/5U alkaline phosphatase) (Beyotime, Suzhou, China) at 37°C for 1 h, and used for immunoprecipitation as described above.
Alkaline phosphatase
Manufactured by Beyotime
Sourced in China
Alkaline phosphatase is an enzyme found in various tissues of the body, including the liver, bones, and kidneys. Its core function is to catalyze the hydrolysis of phosphate esters, playing a role in various metabolic processes.
Automatically generated - may contain errors
Lab products found in correlation
Alp staining kit, by Beyotime (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Alizarin red, by Sangon (1 mentions)
Alizarin red, by Beyotime (1 mentions)
Stempro osteogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Adipogenic induction medium, by ScienCell (1 mentions)
Oil red o, by Solarbio (1 mentions)
Alizarin red s, by Solarbio (1 mentions)
Fitc phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cck 8, by Beyotime (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
P1081, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Beyotime (1 mentions)
Transferrin, by Solarbio (1 mentions)
Lysozyme, by Solarbio (1 mentions)
Proline, by Solarbio (1 mentions)
Papain, by Beyotime (1 mentions)
Kanamycin, by Merck Group (1 mentions)
11 protocols using alkaline phosphatase
1
Alkaline Phosphatase Treatment of Liver Lysates
2
Osteogenic Differentiation of EMSCs
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EMSCs were cultured with the osteogenic induction medium. On days 7 and 21, the cells were stained using an alkaline phosphatase (ALP) staining kit (Beyotime, China) or Alizarin Red (Sangon, China) separately, according to the previous study.23
3
Multilineage Differentiation of Stem Cells
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DFCs, iDFCs and dDFCs were seeded into a six-well plate (1 *105 cells/well) separately and further cultured overnight for eight hours.
To induce osteogenic differentiation, cells were infected with AdBMP9, and the medium was replaced with StemPro Osteogenesis Differentiation Kit (Gibco) for two weeks. Then the differentiated cells were fixed with 4% polyoxymethylene for 20 min, followed by Alkaline phosphatase (Beyotime) staining and Alizarin Red (Beyotime) staining to assess mineral deposition. Cells infected with AdGFP were used as control.
To induce chondrogenic differentiation, cells were infected with AdBMP9, and the medium was replaced with Chondrogesis Differentiation medium (Biowit) for two weeks. The induced cells were fixed with 4% polyoxymethylene for 20 min, followed by alcian blue staining. Cells infected with AdGFP were used as control.
To induce adipogenic differentiation, cells were infected with Ad PPARγ2, and the medium was replaced with Adipogesis Differentiation medium (Biowit) for one week. The differentiated cells were fixed with 4% polyoxymethylene for 20 min and then stained with 0.3% Oil Red O (Sigma) solution to evaluate adipogenesis. Cells infected with AdRFP were used as control.
To induce osteogenic differentiation, cells were infected with AdBMP9, and the medium was replaced with StemPro Osteogenesis Differentiation Kit (Gibco) for two weeks. Then the differentiated cells were fixed with 4% polyoxymethylene for 20 min, followed by Alkaline phosphatase (Beyotime) staining and Alizarin Red (Beyotime) staining to assess mineral deposition. Cells infected with AdGFP were used as control.
To induce chondrogenic differentiation, cells were infected with AdBMP9, and the medium was replaced with Chondrogesis Differentiation medium (Biowit) for two weeks. The induced cells were fixed with 4% polyoxymethylene for 20 min, followed by alcian blue staining. Cells infected with AdGFP were used as control.
To induce adipogenic differentiation, cells were infected with Ad PPARγ2, and the medium was replaced with Adipogesis Differentiation medium (Biowit) for one week. The differentiated cells were fixed with 4% polyoxymethylene for 20 min and then stained with 0.3% Oil Red O (Sigma) solution to evaluate adipogenesis. Cells infected with AdRFP were used as control.
4
Multilineage Differentiation of ADMSCs
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After passage 3 ADMSCs were grown to a confluence of 80% to 90%, the medium was replaced with osteoblast induction medium (ScienCell, USA) or adipogenic induction medium (ScienCell, USA). Alkaline phosphatase (Beyotime, China) staining was performed on the 9th day of induction, and Alizarin red S (Solarbio, China) staining was performed on the 21st day of induction to evaluate osteogenesis. Oil red O (Solarbio, China) staining was performed on the 14th day of induction to evaluate lipid droplet formation. Staining was observed and imaged under a microscope (Olympus Corporation, Japan).
5
Silk Fibroin Scaffold Characterization
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Bombyx mori cocoons were purchased from Jiangsu province, China. CaCl2, sodium alginate and HClwere collected from Aladdin (China). CCK-8, alkaline phosphatase (ALP), and BCA assay kits were ordered from Beyotime Biotechnology Co., Ltd. (Jiangsu, China). FITC-phalloidin and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Gibco (USA). DMEM and 100 U/mL penicillin and streptomycin were purchased from Hyclone (South Logan, USA).
6
Alkaline Phosphatase Treatment of Immunoprecipitated Proteins
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Samples used for the alkaline phosphatase treatment were processed as described for Western blotting, except that after the lysis buffer washes, PBS was added to the immunoprecipitated proteins bound to the beads. Samples were treated with alkaline phosphatase (D7027; Beyotime) in the presence or absence of alkaline phosphatase inhibitor (P1081; Beyotime) and incubated for 1 h at 30°C with occasional shaking. Untreated samples were used as controls. The immunoprecipitated proteins were then washed twice with the wash buffer without protease inhibitors and released from the beads by boiling in SDS loading buffer.
7
Propagation and Titration of Tembusu Virus in DF-1 Cells
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DF-1 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% inactivated fetal calf serum (FCS) in 5% CO2 at 37℃. TMUV JS804 was isolated by our laboratory and propagated in DF-1 cells as previously described [11 (link)]. All the experiments were approved by the ethical committee of Jiangsu Academy of Agricultural Sciences (approval No. SYXK-2015-0020). The titer of virus is calculated as TCID50 (50% tissue culture infective dose) according to the Reed-Muench method [26 ]. In brief, Serial 10-fold dilutions of the stock virus were inoculated into a monolayer of DF-1. After two-hour incubation at 37℃, inoculum was removed and cells were overlaid with a mixture of 1% agarose and cell culture media. Plaque formation was continuously observed daily for four days and TCID50 was calculated according to the method of Reed-Muench. TMUV-monoclonal antibody was generated and maintained by our laboratory as previously described [8 (link)]. Alkaline phosphatase and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG were purchased from the Beyotime Institute of Biotechnology (China). Anti-HSPA9 (ab171089, mouse) was purchased from Abcam (UK).
8
Enzymatic Activity Assays Optimization
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β-Galactosidase (β-Gal), horseradish peroxidase, alkaline phosphatase, papain, pepsin, and α-mannosidase were purchased from Beyotime (Shanghai, China). Lysozyme, glucose, bovine serum albumin, γ-globulin, transferrin, mannitol tryptophan and proline were purchased from Solarbio (Beijing, China). Ciprofloxacin, ampicillin, kanamycin, dimethyl sulfoxide (DMSO), Polypropylene pyrrolidone K30 (PVP-K30, MW = 40,000) and all other chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, United States). Luria-Bertani broth (LB) medium was purchased from BD Biosciences (San Jose, CA). All aqueous solutions were prepared with Milli-Q water (≥18 MΩ, Milli-Q, Millipore).
9
Differentiation of Human iPSCs into MSCs
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In accordance with our previous study (Hu et al., 2015), the human iPSC line (IPS-S cell line, RRID: CVCL_C876) was provided by the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Liao et al., 2008), and cultured and expanded on human ESC-Qualified BD Matrigel (BD Biosciences, Sparks, MD, USA)-coated plates in mTESR1 (StemCell Technologies, Vancouver, BC, Canada). iPSCs were identified by immunofluorescent staining of Nanog, OCT4, SSEA4, TRA-1-81, and alkaline phosphatase (Beyotime Biotechnology, Shanghai, China, Cat# C3206) (Hu et al., 2020). When the iPSCs reached 80–90% confluence, mTESR1 was replaced with Dulbecco’s modified Eagle medium-low glucose (Corning, Tewksbury, MA, USA) containing 10% (vol/vol) fetal bovine serum (Life Technologies, Grand Island, NY, USA) and 2 mM L-glutamine. After 14 days of culture, the cells were serially trypsinized (0.25% trypsin/1 mM ethylene diamine tetraacetie acid; Life Technologies) and reseeded three times. Cells at passage 4 generally had a morphology resembling MSCs and were used for identification and further experiments.
10
Osteogenesis Evaluation of H2O2 and Corynoline
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The cells were seeded at 5 × 104 cells/well in a 24‐well plate. After being treated with H2O2 and Corynoline, the cells were cultured in DMEM with 10 mm of β‐glycerol phosphate and 20‐μM ascorbic acid. The media were freshened every other day. The ALP activity was detected using alkaline phosphatase (ALP) staining kit (Beyotime) seven days after differentiation. The cells were then cultured for three weeks under osteogenic conditions, fixed, and stained with alizarin red (ARS) (Beyotime).
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