Mesenchymal stem cells were derived from the adipose tissue (AD-MSCs) of a three-month-old female Polish Large White pig. Tissue sample collection was approved by the Local Ethical Commission for Experiments on Animals at Poznan University of Life Sciences, Poznan, Poland (approval no. 57/2012). Following Stachecka et al. [39 (link)], the AD-MSCs were cultured in Advanced DMEM (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS (v/v) (Sigma-Aldrich, St. Louis, MO, USA), 5 ng/mL FGF-2 (PromoCell GmbH, Heidelberg Germany), 2 mM L-glutamine (Gibco), 1 mM 2-mercaptoethanol (Sigma-Aldrich), 1 × antibiotic antimycotic solution (Sigma-Aldrich), and 1 × MEM NEAA (Gibco) at 37 °C in 5% CO2. To avoid the possible influence of FBS-derived miRNAs on obtained results, the same part of filtered FBS was used during the whole cell culture experiment. The AD-MSCs were propagated by passaging using standard cell culture procedures, and their stemness was confirmed by staining for positive (CD44, CD90, CD105) and negative (CD45) markers (Abcam, Cambridge, UK).
Fgf 2
Manufactured by PromoCell
Sourced in United States, United Kingdom
FGF-2 is a recombinant protein that represents the basic fibroblast growth factor. It is a heparin-binding growth factor that is involved in a variety of biological processes, including cell growth, cell differentiation, tissue repair, and angiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Advanced dmem, by Thermo Fisher Scientific (2 mentions)
Mem neaa, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Antibiotic antimycotic solution, by Merck Group (2 mentions)
Hdf strains, by Thermo Fisher Scientific (2 mentions)
Tgfβ1, by PromoCell (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions)
C 12200, by PromoCell (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
7 protocols using fgf 2
1
Adipose-Derived Mesenchymal Stem Cell Culture
2
Adipogenic Differentiation of MSCs
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Adipogenesis was induced by culturing early-passage MSCs in an adipogenic differentiation medium composed of Advanced DMEM (Gibco), 10% FBS (Sigma-Aldrich), 1 × antibiotic antimycotic solution (Sigma-Aldrich), 1 × MEM NEAA (Gibco), 5 ng/mL FGF-2 (PromoCell GmbH), 1 × linoleic acid albumin, 1 × ITS, 1 µm dexamethasone (Sigma-Aldrich), 100 µm indomethacin (Sigma-Aldrich), and 50 mM IBMX (Sigma-Aldrich). The cells were cultured for ten days. Adipogenic differentiation was monitored using visual examination of lipid droplet formation under a phase-contrast microscope (Nikon TS100 Eclipse, Melville, NY, USA) and BODIPY staining. Cells were fixed with 4% paraformaldehyde in PBS (w/v) for ten minutes at room temperature and washed thrice with PBS. The cells were then incubated with BODIPY 493/503 (Thermo Fisher, Waltham, MA, USA) in PBS (3 µg/mL) and washed thrice in PBS. The nuclei were counterstained with DAPI in Vectashield medium (Vector Laboratories, Newark, CA, USA) and examined under a fluorescence microscope (Nikon E600 Eclipse, Melville, NY, USA). Each measurement was performed in triplicate.
3
Expansion and Maintenance of Adipose-Derived Stromal Cells
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For cell expansion, freshly isolated SVF cells were plated at a density of 1 × 105 cells/cm2. When confluency was achieved, one part of the cells were either detached with 0.05% trypsin/0.01% EDTA (#25300054, Gibco) and re-plated at a density of 1 × 105 cells/cm2 (ASCs). The other part of the cells was further cultured without passaging for 28 days (Unpass cells). Cells were cultured in complete medium (CM), consisting of alpha-minimal essential medium (α-MEM, #12571063, Gibco) supplemented with 10% fetal bovine serum (FBS, #16000044, Gibco), 1% HEPES (#15630080, Gibco), 1% sodium pyruvate (#11360070, Gibco), and 1% penicillin-streptomycin-glutamine (PSG, #10378016, Gibco) solution, supplemented with 5 ng/mL fibroblast growth factor-2 (FGF-2, #233-FB,R&D systems) and filtered through a 100 µm strainer (#352360, BD Falcon). Human umbilical vein endothelial cells (HUVECs) (#C-12200, PromoCell) were cultured on 2% gelatin-coated plates directly after thawing, within CM medium also supplemented with 5 ng/mL FGF-2. Cells were cultured at 37°C in a 5% CO2 with a 95% air-humidified incubator.
4
Fibroblast Culture and Maintenance Protocol
5
Maintaining Human Dermal Fibroblasts
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Three HDF strains (Invitrogen, Paisley, UK), passages 3–15, were used throughout. HDFs were maintained at 37 °C in a humidified atmosphere of 5% CO2, as subconfluent monolayers in fibroblast complete medium: Dulbecco's modified Eagle's medium, 0.5% antibiotic solution, and 10% fetal bovine serum (Invitrogen). Sometimes serum-free medium (SFM) was used. All drugs were purchased from Tocris (Bristol, UK), except FGF2 and TGFβ1 (Promocell, Heidelberg, Germany).
6
Cultivation of Human Dermal and Mammary Fibroblasts
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Normal human dermal fibroblasts (NHDFs) and Fibroblast Growth Medium containing 2% fetal calf serum (FGM), insulin (5μg/mL) and FGF-2 (1ng/mL) were obtained from PromoCell (http://www.promocell.com/ ) and cells were cultured as specified by the supplier. Human Primary Mammary Fibroblasts (HPMFs), HPMF growth media (HPMF-GM), the Fibroblast Medium Supplement Kit (FBS, hydrocortisone, L-glutamine, FGF and an antibiotic-anti-mycotic solution) and gelatin coating solution were obtained from Cell Biologics (http://www.cellbiologics.net ). HPMF cells were cultured in HPMF-GM with the added supplements for 6–7 passages as specified by the supplier. MDA-MB-231 breast cancer cells were obtained from ATCC and cultured for 12–15 passages in MCF media. MCF medium is DMEM/F12 (Life Technologies, Carlsbad, CA) supplemented with 5% horse serum, human recombinant EGF, insulin, hydrocortisone and cholera toxin, all obtained from Sigma Aldrich (St. Louis, MO), and penicillin-streptomycin from Thermo -Fisher (Grand Island, NY) [21 (link)]. GFP tagged human dermal fibroblasts (NHDFs-GFP) were obtained from Angio-proteomie (www.angioproteomie.com ) and cultured according to the supplier’s directions.
7
Culturing Human Endothelial Cells
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Immortalized human umbilical vein ECs (CI-huVECs, InSCREENeX, Braunschweig, Germany) and human cerebral microvascular ECs (hCMEC/D3; Merck Millipore, Darmstadt, Germany) were cultured at 37°C and 5% of CO 2 in EC growth medium (ECGM, PromoCell, Heidelberg, Germany) supplemented with 10% of fetal calf serum (FCS, Thermo Fisher Scientific, Waltham, MA, USA) or in EndoGRO-MV complete medium (Merck Millipore) supplemented with 1 ng/mL of FGF-2 (PromoCell) and 5% of FCS, respectively. For rescue experiments, human plasma fibronectin (F2006, Sigma-Aldrich, St. Louis, MO, USA), human cellular fibronectin (F2518, Sigma-Aldrich), a proteolytic human plasma fibronectin 70 kD fragment (F0287, Sigma-Aldrich), a human fibronectin 120 kD cell attachment fragment (Part Number 175, YO Proteins, Ronninge, Sweden), human type IV collagen (C5533, Sigma-Aldrich), recombinant periostin (rPOSTN; RPH339Hu01, Cloud-Clone Corp., Katy, TX, USA), and recombinant fibulin-5 (rFBLN5; 9006-FB-050, R&D Systems, Minneapolis, MN, USA) were either supplemented to the ECGM or used to coat cell culture plates with the indicated concentrations. If not stated otherwise, the cells were cultured for 48 hours with ECM protein supplementation. Cells cultured on uncoated, tissue culture treated plates served as controls.
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