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).
Fabp4
Manufactured by R&D Systems
Sourced in United States
FABP4, also known as Adipocyte Fatty Acid-Binding Protein, is a laboratory research product that functions as a fatty acid-binding protein. It is involved in the intracellular transport and metabolism of fatty acids.
Automatically generated - may contain errors
Lab products found in correlation
Osteocalcin, by R&D Systems (4 mentions)
Oil red o, by Polysciences (3 mentions)
Alkaline phosphatase alp, by Merck Group (3 mentions)
Fgf21, by R&D Systems (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
C ebpα, by Abcam (1 mentions)
Pparγ, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
Osteopontin, by Abcam (1 mentions)
Enhanced chemiluminescence detection system, by Cytiva (1 mentions)
Adiponectin, by Abcam (1 mentions)
β actin, by R&D Systems (1 mentions)
Adiponectin, by R&D Systems (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Leptin, by R&D Systems (1 mentions)
Fabp4, by BioVendor (1 mentions)
Aggrecan, by R&D Systems (1 mentions)
13 protocols using fabp4
1
Differentiation of hDPSCs Into Adipocytes and Osteocytes
2
Protein Extraction and Western Blot Analysis
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Cell pellets were lysed in lysis buffer NETN (20 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.5% NP-40, protease inhibitor tablet (Roche), 0.5 mM DTT). Samples were incubated in the cold room for 30 min followed centrifugation at 4°C with maximum speed for 10 min. The supernatant was collected and the concentration determined by BCA quantification (Pierce BCA protein Assay kit, Cat.23225, Thermo scientific) to allow normalization between samples for western blotting. Samples were mixed with Laemmli Sample Buffer (4×) (containing 1.0 M Tris-pH 6.8, 8% SDS, glycerol, β-mercaptoethanol (10%), bromophenol blue) for boiling at 100°C for 10 min. The samples were then separated by SDS-PAGE and analyzed by standard immunoblotting using running buffer from Invitrogen (NuPAGE™ Tris-acetate SDS running buffer (20×), LA0041) and transfer buffer from Thermofisher (NUPAGE transfer buffer, NP00061). The blotting processes was performed as previous described (27 (link)). Antibodies used for Western blot are the following: ATRX (Santa Cruz, sc-15408, 1:800), FABP4 (R&D, AF1443, 1:5000), C/EBPα (CST, #2295, 1:1000), PPARγ (CST, #2430), β-actin (Abcam, ab8224), GAPDH (Abcam, ab8245, 1:1000).
3
Western Blot Analysis of Adipogenic Markers
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The BM-MSC treated with induction medium or untreated cells were homogenized in lysis buffer [10 mM Tris-Cl (pH 7.5), 50 mM NaCl, and 1% Triton-X-100 containing phenylmethylsulfonyl fluoride (1 mM) and protease inhibitor cocktail] and centrifuged at 12,000 xg for 15 min at 4°C and the supernatant was estimated for protein content. 100 μg protein of each sample was subjected to 6% SDS-PAGE and electrotransferred onto nitrocellulose membrane. The membranes were incubated with antibodies against adiponectin (Abcam), FABP4 (R&D Systems; http://www.rndsystems.com/ ), osteopontin (Abcam, http://www.abcam.com/ ), and β-actin (R&D Systems) followed by incubation with HRP-conjugated corresponding secondary antibodies. The signals were detected using an enhanced chemiluminescence detection system (Amersham Biosciences, http://www.gelifesciences.com/ ).
4
Protein Expression Analysis by Western Blot
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Western blot analysis was performed as previously described [29 (link)]. Primary antibodies included SLC25A28 (Abcam), adiponectin (R&D), fibroblast growth factor 21 (FGF21) (R&D), fatty acid binding protein 4 (FABP4) (R&D), UCP-1 (R&D), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (Abways), ATGL (Abmart), perilipin 1 (Abways), perilipin 2 (Abways), leptin (R&D), and β-actin (Santa Cruz).
5
Measuring Adiponectin, FABP4 and FGF21 Levels
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Concentrations of adiponectin, FABP4 and FGF21 were measured using enzyme‐linked immunosorbent assays kits for adiponectin (R&D Systems, Minneapolis, MN, USA), FABP4 (Biovendor, Modrice, Czech Republic) and FGF21 (R&D Systems), respectively. Variables of liver function, renal function, glucose and lipid metabolism were measured as previously described7 . Homeostasis model assessment of insulin resistance (HOMA‐IR) was calculated as insulin (μU/mL) × glucose (mg/dL) / 405.
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
Multilineage Differentiation of iPSCs
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To induce neural differentiation, PDGFRβ+ iPC clusters were incubated on poly-l -lysine-coated glass coverslips for 7 days in neurobasal medium (Invitrogen, Carlsbad, CA, USA) supplemented with B-27 (Invitrogen) and all-trans retinoic acid (0.2 μM; Sigma, St. Louis, MO, USA) [20 (link)]. Differentiated cells were labeled with antibodies against Iba1 (Abcam), CD11b (BD Pharmingen), major histocompatibility complex (MHC) class 1 (Abcam), and Tuj1 (Stem Cell Technologies, Vancouver, BC, Canada). To induce osteoblastic or adipogenic differentiation, PDGFRβ+ iPCs were cultured in osteogenic or adipogenic differentiation medium, respectively, according to the manufacturer’s protocol (SC 010; R&D systems, Minneapolis, MN, USA). The resulting differentiated cells were labeled with antibodies against osteopontin (Santa Cruz Biotechnology) or fatty acid binding protein 4 (FABP4) (R&D Systems), respectively. Alternatively, PDGFRβ+ iPCs incubated in adipogenic differentiation medium were stained with the lipid-specific dye Oil Red O as described previously [32 (link)].
8
Isolation and Adipogenic Differentiation of Preadipocytes
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Stromal vascular fraction from epididymal white adipose tissue was isolated using collagenase type VIII (Sigma) digestion. Isolated cells were stained (0.25-0.5 μg/ml per 106 cell in 100 μl volume) with CD31-PE (BD Biosciences cat# 560238, San Jose, CA), CD34-FITC (BD Biosciences cat# 553373), CD45-APC-Cy7 (Biolegend cat# 103116, San Diego, CA) and CD140a-BV421 (BD Pharmingen cat# 562774) and cell sorted with FACS Aria and analyzed with FACS Diva for CD31−, CD34+, CD45− and CD140a+ preadipocytes [16 (link)]. Cells were then plated and cultured according to the adipogenic differentiation of mouse mesenchymal stem cells protocol (R&D Systems, Minneapolis, MN). After differentiation, adipocytes were fixed with 4% paraformaldehyde and stained with primary antibody Fabp4 (R&D Systems cat# AF1443, Minneapolis, MN), secondary antibody α-goat Alexa Fluor-488 (ABCAM cat# ab150129, Cambridge, MA), HCS LipidTOX Red neutral lipid stain (Life Technologies, Carlsbad, CA) and Vectashield with DAPI (Vector Laboratories cat# H-1200, Burlingame, CA). Confocal microscopy was performed using Axiovert 200M (Zeiss) with 63x/1.4 oil objective and images analyzed with Zeiss LSM510 software.
9
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] .
10
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).
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