Adipogenic and osteogenic differentiation was performed according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). To detect adipogenic differentiation, hDPSCs were stained with Oil Red O (Polysciences, Warrington, PA, USA) and fatty acid-binding protein-4 (FABP-4; R&D Systems). Osteogenic differentiation detection was performed by staining hDPSCs with alkaline phosphatase (ALP; Millipore, Darmstadt, Germany) and osteocalcin (R&D Systems).
Osteocalcin
Manufactured by R&D Systems
Sourced in United States
Osteocalcin is a protein produced by osteoblasts, the cells responsible for bone formation. It is a key component of the bone extracellular matrix and plays a role in the mineralization and regulation of bone metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Oil red o, by Polysciences (4 mentions)
Fabp4, by R&D Systems (4 mentions)
Alkaline phosphatase alp, by Merck Group (3 mentions)
Quantifluor dsdna system, by Promega (2 mentions)
Aggrecan, by R&D Systems (2 mentions)
Estradiol, by R&D Systems (1 mentions)
Elisa kits to detect, by R&D Systems (1 mentions)
Igfbp3, by R&D Systems (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Interleukin 10, by Thermo Fisher Scientific (1 mentions)
Osteopontin, by R&D Systems (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Elisa kit, by Nanjing Jiancheng (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Santa Cruz Biotechnology (1 mentions)
Fv10i confocal system, by Olympus (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Macsquantifytm, by Miltenyi Biotec (1 mentions)
10 protocols using osteocalcin
1
Differentiation of hDPSCs Into Adipocytes and Osteocytes
2
Quantification of Hormones and Enzymes
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ELISA kits to detect alkaline phosphatase (ALP) activity and the concentrations of estradiol, osteocalcin, IGF-1, IGFBP-3, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were purchased from R&D Systems (Minneapolis, USA).
3
Osteoblast Response to Extracellular Vesicles
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MG63 osteoblast-like cells were cultured as described. After reaching confluence, culture media were replaced with fresh DMEM with 10% FBS without pen-strep and cells were treated with 5 µg/mL (based on protein content) of MVs (either GM or OM as described above) or EXs for 24 or 48 h. At the time of harvest, conditioned media were collected, cells were washed with 1 mL of phosphate-buffered saline (PBS), and lysed with 500 µL 0.05% Triton X-100 in PBS per well. After a single freeze-thaw, cells were sonicated. From the cell lysate, DNA quantification and alkaline phosphatase-specific activity were assessed using Quantifluor dsDNA system (Promega, Madison, WI, USA) and via the formation of para-nitrophenol from para-nitrophenylphosphate at pH 10.2, respectively. Osteocalcin (R&D, Minneapolis, MN, USA), osteopontin (R&D Systems, Minneapolis, MN, USA), vascular endothelial growth factor (R&D Systems), interleukin-6 (Peprotech, Cranbury, NJ, USA), and interleukin-10 (Peprotech, Cranbury, NJ, USA) levels were measured from the conditioned media using enzyme-linked immunoassay (ELISA) following the manufacturer’s instructions.
4
Multilineage Differentiation of DPSCs
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Differentiation into adipocytes, osteoblasts and chondrocytes of cultured DPSCs was carried out according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). For the detection of adipogenic differentiation, the cells were stained with oil red O (Polysciences Inc., Warrington, PA, USA) and the fatty acid‐binding protein‐4 (R&D Systems). For the detection of osteogenic differentiation, the cells were stained with osteocalcin (R&D Systems). Calcification of osteogenic monolayers was also visualized using Alizarin Red S (Merck, Darmstadt, Germany). For the detection of chondrogenic differentiation, the cells were stained with aggrecan (R&D Systems), known as the major proteoglycan in the articular cartilage.
5
Serum Bone Marker Analysis in Mice
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Non-fasting blood samples were obtained from all mice before sacrifice and stored at −20°C to evaluate the expression of serum bone biochemical markers. The serum bone biochemical markers were detected with the method of enzyme-linked immunosorbent assay (ELISA) kit. The ELISA kit of ALP, procollagen type 1 amino terminal propeptide (P1NP), C-terminal cross-linked telopeptide of type-I collagen (CTX) and osteocalcin (OCN) were provided by Nanjing Jiancheng Bioengineering Institute (China), with osteocalcin (OCN) by R&D Systems (USA) and HbA1C by Hangzhou Jianglai Biotechnology Co. (China).
6
Multilineage Differentiation of DPSCs
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DPSCs were differentiated into adipocytes, osteoblasts, or chondrocytes using adipogenic osteogenic or chondrogenic differentiation-inducing medium (Lonza, Basel, Switzerland) according to the manufacturer’s instructions. Cells were stained with Oil red O (Polysciences, Warrington, PA) and fatty acid-binding protein-4 (FABP-4; R & D Systems, Minneapolis, MN) to assess adipogenic differentiation. Cells were stained with alkaline phosphatase (ALP; Millipore, Billerica, MA) and osteocalcin (R & D Systems) to assess osteogenic differentiation. Cells were stained with aggrecan (R & D Systems) to assess chondrogenic differentiation. For the detection of nuclei, cells were stained with 4′-6-Diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO).
7
DPSC Transplantation and Differentiation in Diabetic Rats
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To investigate the survival and the differentiation of transplanted DPSCs, DPSCs isolated from GFP rats were transplanted into the hindlimb skeletal muscles of diabetic rats. Four weeks after the transplantation, the DPSC-transplanted skeletal muscles were excised and fixed in 4 % paraformaldehyde. The specimens were embedded within an OCT compound (Sakura Finetechnical, Tokyo, Japan) and cut into 5-μm sections. The sections were stained with the primary antibody against PECAM-1 (1:400), FABP (1:100), osteocalcin (1:100) (R&D Systems, Minneapolis, MN, USA), neuronal nuclei (NeuN) (1:400; Millipore, Billerica, MA, USA), and glial fibrillary acidic protein (1:400; Santa Cruz). After washing, the sections were incubated with the Alexa fluor 594-conjugated secondary antibodies. DAPI was detected as cell nuclei. Sections were observed under a fluorescence microscope FV10i confocal system (Olympus, Tokyo, Japan).
8
Characterization and Differentiation of DPSCs
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DPSCs at passage 3 were characterized by fluorescence-activated cell sorting (FACS) (FACS Calibur, Becton Dickinson) and then incubated with anti-rat fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibodies against CD49d (#557457; Becton Dickinson) and CD90 (#554894; Becton Dickinson), anti-rat FITC-conjugated hamster monoclonal antibodies against CD29 (#561796; Becton Dickinson), and anti-rat r-phycoerythrin (R-PE)-conjugated mouse monoclonal antibodies against CD34 (#551387; Becton Dickinson) and CD45 (#554878; Becton Dickinson). Isotype-identical antibodies served as controls. Data were analyzed using MACS QUANT software (MACS QuantifyTM, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) [42] .
After 21 days of culture, differentiation was induced using osteogenic or adipogenic differentiation induction medium (Lonza Group AG, Basel, Switzerland). Differentiated osteoblasts were stained with alkaline phosphatase (ALP; MilliporeSigma, Burlington, MA, USA) and osteocalcin (R&D Systems, Inc., Minneapolis, MN, USA). Differentiated adipocytes were stained with Oil Red O (Polysciences, Inc., Warrington, PA, USA) and fatty acid-binding protein-4 (FABP-4; R&D Systems) in accordance with the manufacturer's instructions [43] .
After 21 days of culture, differentiation was induced using osteogenic or adipogenic differentiation induction medium (Lonza Group AG, Basel, Switzerland). Differentiated osteoblasts were stained with alkaline phosphatase (ALP; MilliporeSigma, Burlington, MA, USA) and osteocalcin (R&D Systems, Inc., Minneapolis, MN, USA). Differentiated adipocytes were stained with Oil Red O (Polysciences, Inc., Warrington, PA, USA) and fatty acid-binding protein-4 (FABP-4; R&D Systems) in accordance with the manufacturer's instructions [43] .
9
Mesenchymal Stem Cell Differentiation Protocol
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The human mesenchymal stem cell functional identification kit (R&D systems, Minneapolis, MN) was employed for the differentiation of hMSCs into adipocytes and osteoblasts. Briefly, hMSCs were cultured in minimum essential medium (MEM, Gibco, Carlsbad, CA) containing adipogenic and osteogenic supplements for 21 days to induce differentiation into adipocytes and osteoblasts respectively. The medium was replaced with fresh medium every 3–4 days. After 21 days, differentiated cells were fixed with 4% paraformaldehyde and incubated with a primary antibody against fatty acid binding protein 4 (FABP4, 10 ug/ml, R&D Systems, Minneapolis, MN) for adipocytes and osteocalcin (10 ug/ml, R&D Systems, Minneapolis, MN) for osteoblasts. The cells were washed and incubated with fluorescein-labeled anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). Stained cells were observed using a microscope (Nikon, Tokyo, Japan).
10
Quantifying Osteogenic Factors In Cell Lysates
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