Decellularized matrix and beta‐tricalcium phosphate (β‐TCP) were provided by University of Shanghai for Science and Technology. Cell counting kit‐8 (CCK‐8) kit, alkaline phosphatase (ALP) kit, bicinchoninic acid (BCA) assay kit, and 4′,6‐diamidino‐2‐phenylindole (DAPI) were obtained from Beyotime Biotechnology Co (Shanghai, China). Phalloidin was obtained from Invitrogen Co. (USA). The Runx‐2, OCN, OPN, β‐actin antibodies were obtained from CST (MA, USA). Alexa Fluor 488‐labeled and Alexa Fluor588‐labeled Goat Anti‐Rabbit IgG (H+Lx) secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).
Alkaline phosphatase alp kit
Manufactured by Beyotime
Sourced in China
The Alkaline phosphatase (ALP) kit is a laboratory product used for the quantitative determination of ALP activity in various biological samples, such as serum, plasma, or cell lysates. ALP is an enzyme involved in the hydrolysis of phosphate esters, and its activity is often measured as an indicator of certain physiological or pathological conditions.
Automatically generated - may contain errors
Lab products found in correlation
Ars kit, by Beyotime (2 mentions)
Triton x 100, by Beyotime (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexafluor488 labeled, by Jackson ImmunoResearch (1 mentions)
Bicinchoninic acid bca assay kit, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Cell counting kit 8 cck 8 kit, by Beyotime (1 mentions)
Alizarin red staining kit, by Beyotime (1 mentions)
Malachite green phosphate detection kit, by Beyotime (1 mentions)
Alp assay kit, by Beyotime (1 mentions)
β glycerophosphate disodium, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Hematoxylin eosin h e stain, by Thermo Fisher Scientific (1 mentions)
Perfect real time rt reagent kit, by Takara Bio (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Eastep super total rna extraction kit, by Promega (1 mentions)
11 protocols using alkaline phosphatase alp kit
1
Decellularized Matrix and β-TCP for Bone Regeneration
2
Osteogenic Differentiation Assays
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Magnesium chloride (MgCl2), gallic acid, potassium hydroxide (KOH), dopamine hydrochloride, Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), ethylene diamine tetraacetic acid (EDTA), potassium chloride (KCl), l -ascorbic acid, sodium hydroxide (NaOH), glycine (Gly) and calconcarboxylic acid were obtained from Aladdin Bio-chem Technology (Shanghai, China). Cell counting kit-8 (CCK-8), collagen type Ⅰ (Col-Ⅰ) and adenosine triphosphate (ATP) assay kit were purchased from Solarbio (Beijing, China). Alkaline phosphatase (ALP) kit, malachite green phosphate detection kit, alizarin red staining kit, live and dead bacterial staining kit were obtained from Beyotime Biotechnology Co. (Jiangsu, China).
3
Osteogenic Differentiation of rBMSCs
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The 1×105 rBMSCs were seeded in 6-well plates and co-cultured with microsponges using the Transwell apparatus. After 14 days of culturing in an osteogenic medium, each group was fixed with 4% paraformaldehyde at room temperature for 30 min and washed twice using PBS, then incubated with an alkaline phosphatase (ALP) kit (Beyotime, China) in the dark for 2 h. The cells were washed three times with PBS and observed under the OPM. Furthermore, an ALP assay kit (Beyotime, China) was used to analyze the ALP activity following the manufacturer’s instructions. OD was measured at 405 nm using a microplate reader.
4
Osteogenic Differentiation of Mesenchymal Stem Cells
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Human bone marrow mesenchyme stem cells were seeded at 12‐well culture plates and grew to confluence with MSCM medium. Then, the MSCM medium was removed, and osteogenic induction medium composed of MEM containing 10% FBS, 10 mmol/L β‐glycerophosphate disodium (Sigma, G9422), 0.2 mmol/L ascorbic acid (Sigma, A5960), 0.1 µmol/L dexamethasone (Sigma, D1756)26 was added to induce osteogenic differentiation. CHS and Si ion were added in osteogenic induction medium, and HBMSCs were cultured in these media for 7 days. Then, alkaline phosphatase (ALP) kit purchased from Beyotime Biotechnology, Shanghai (P0321), was used to stain the cells to evaluate the ALP protein synthesis in the HBMSCs cultured with different media.
5
Poloxamer 407 Enhances Rabbit BMSC Osteogenesis
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Poloxamer 407 was supplied by Bayee Chemical Co., Ltd. (Hangzhou, China). Low glucose Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), streptomycin–penicillin, and 0.25% trypsin EDTA were obtained from Gibco® Life Technologies (CA, United States). For cell experiments, the rabbit BMSCs were purchased from ATCC Co., Ltd. (Maryland, United States). Enzyme linked immunosorbent assay (ELISA) Kits of TGF-β was supplied by ZCIBIO Technology Co.,Ltd. (Shanghai, China) and VEGF was obtained from Hengyuan Biotechnology Co., Ltd. (Shanghai, China). Calcein-AM/Propidium Iodide (PI), Cell Counting Kit-8 (CCK-8) assay, BCA Protein Assay Kit, Alkaline Phosphatase (ALP) Kit, and RIPA Lysis Buffer were purchased from Beyotime Biotechnology (Shanghai, China). Osteogenic induction medium of rabbit BMSCs and stain of Alizarin Red were supplied by Cyagen Biosciences (CA, United States). Eastep Super Total RNA Extraction Kit was supplied by Promega (Shanghai, China). Perfect Real Time RT reagent kit was obtained from Takara Bio (Dalian, China). For histological staining, hematoxylin eosin (H&E) stain was purchased from Thermo Fisher Scientific (MA, United States). For immunofluorescence staining, primary antibodies were supplied by Abcam (Cambridge, United Kingdom), and secondaries antibodies were supplied by Jackson ImmunoResearch Laboratories (West Grove, PA, United States).
6
Mussel-Derived Peptides for Bone Implant Optimization
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The clickable mussel-derived peptides were prepared using standard Fmoc solid-phase synthesis47 (link). Quartz wafers (10 mm or 15 mm in diameter) with 80–100 nm TiO2 layer were prepared by the key Laboratory of Advanced Technologies of Materials, Southwest Jiaotong University (Chengdu, China). For in vivo experiments, pure Ti screws (2 mm × 10 mm) were purchased from Tianjin Zhengtian Medical Device Company (Tianjin, China). Cell counting kit-8 (CCK-8) kit, a Live/Dead cell staining kit and FITC-labeled Phalloidin staining were purchased from Yeasen Biotechnology Co. (Shanghai, China). Alkaline phosphatase (ALP) kit, ARS kit and Triton X-100 were obtained from Beyotime Biotechnology Co. (Jiangsu, China). The other chemical reagents or antibodies unless mentioned elsewhere were almost purchased from Sigma, Abcam or Invitrogen.
7
Quantifying Osteoblastic Differentiation in 3D
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The MC3T3-E1 cell line was inoculated in the scaffold and cultured for 7 days and 14 days [26 (link)]. The medium of each well was removed and washed three times with PBS to remove unattached cells. The cells were lysed by adding 0.5% Triton-X 100 cell lysate to each well, and the sample was obtained after high-speed centrifugation. The total protein content of each well was measured using a BCA protein concentration assay kit (Solarbio, Beijing, China), and a standard curve was prepared. The OD value was measured at 562 nm using a microplate reader. The intracellular ALP content was measured using an alkaline phosphatase (ALP) kit (Beyotime, Shanghai, China), and the OD value was measured at 520 nm using a microplate reader. The ALP data were normalized by the total amount of protein, as determined by the BCA Protein Concentration Kit.
8
Hydrogel Functionalization for Cell Culture
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HA sodium salt from Streptococcus Equi was supplied by Aladdin Co., Ltd. (Shanghai, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), cysteine, and dithiothreitol (DTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol diacrylate (PEGDA, MW 3400 Da) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Alkaline phosphatase (ALP) kit, ARS kit and Triton X-100 were obtained from Beyotime Biotechnology Co. (Jiangsu, China). The Cell Counting Kit-8 (CCK-8) kit was purchased from Thermo Fisher Scientific (Carlsbad, USA), and the live/dead cell staining kit was purchased from Bioss Biotech (Bioss, china). The Other reagents or antibodies, unless specifically mentioned elsewhere, were purchased from Affinity Biosciences, Abcam, Sigma, or GeneTex.
9
Scaffold-Mediated BMSC Differentiation
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The effect of scaffolds at the later stage of GW2580 release on proliferation, adhesion, and osteogenic differentiation of mouse BMSCs (mBMSCs) was analyzed. The CPC, CPC/hydrogel, and CPC/hydrogel/GW2580 scaffolds were put separately into a 24 well culture plate (each in a well) and incubated with 1 mL of standard culture medium. The medium was refreshed daily. After 7 days of incubation, scaffolds were collected for following study. mBMSCs were isolated as previous report [36 ]. 1 × 104 mBMSCs were seeded onto each scaffold in a 24-well plate and cultured for 24 and 72 h. Cell viability was then assayed with a CCK-8 kit to test the effect of scaffolds on cell proliferation. Cells cultured in standard medium were used as controls.
To assay cell adhesion on the scaffolds, mBMSCs were evenly seeded onto each scaffold (1 × 104 per scaffold) and co-cultured for 3 days; cell morphology on the scaffolds was detected with a confocal laser scanning microscopy (Zeiss) after fixing, staining with rhodamine-phalloidin and DAPI. To assess the effect of scaffolds on cell function, mBMSCs were evenly seeded into a 24-well plates (5 × 104 per well), cultured with the scaffold extract medium separately for seven days. Then, the cells were fixed, stained with an alkaline phosphatase (ALP) kit (Beyotime, Shanghai, China), and observed under an optical microscope (Leica).
To assay cell adhesion on the scaffolds, mBMSCs were evenly seeded onto each scaffold (1 × 104 per scaffold) and co-cultured for 3 days; cell morphology on the scaffolds was detected with a confocal laser scanning microscopy (Zeiss) after fixing, staining with rhodamine-phalloidin and DAPI. To assess the effect of scaffolds on cell function, mBMSCs were evenly seeded into a 24-well plates (5 × 104 per well), cultured with the scaffold extract medium separately for seven days. Then, the cells were fixed, stained with an alkaline phosphatase (ALP) kit (Beyotime, Shanghai, China), and observed under an optical microscope (Leica).
10
Synthesis and Characterization of Bioactive Polymer Scaffolds
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The chemical reagents involved in our experiments are listed as follows: glycidyl methacrylate (GMA, Mw 142.2) was purchased from J&K company. N-isopropylacrylamide (NIPAm, Mw, 113.2), copper(I)bromide (CuBr, Mw 143.5), 2,2′-bipyridyl (bPy, Mw 156.2), ω-mercaptoundecyl bromoisobutyrate (MUDBr), sodium azide (NaN3, Mw 65.01), N,N-dimethyl formamide (DMF, Mw 73.1), l -ascorbic acid (VC, Mw 176.1) and gelatin were all purchased from Sigma-Aldrich. Methanol (Mw 32.05) and dichloromethane (Mw 84.93) were purchased from Duksan Ltd. Toluene (Mw 92.1), acetone (Mw 58.1), ethanol (Mw 46.1) and 2-propanol (Mw 60.1) were all purchased from VWR-BDH Co. Ltd. Phalloidin-Rhodamine, DAPI and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) were purchased from Sigma-Aldrich. Alkaline phosphatase (ALP) Kit and Bicinchoninic Acid (BCA) Kit were from Beyotime Bio Company, while the BMP-2 ELISA kit (QuantiCyto®) was purchased from Neobioscience Co. Ltd. All cell-culture related reagents were purchased from Gibco Co. Ltd.
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