Fibroblast growth factor (fgf)

Manufactured by R&D Systems

Sourced in United States, Germany

FGF (Fibroblast Growth Factor) is a family of proteins that play a crucial role in cell growth, differentiation, and angiogenesis. This lab equipment is designed to facilitate research and analysis of FGF and its functions.

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Lab products found in correlation

94 protocols using fibroblast growth factor (fgf)

mESC and hESC Culture Protocols

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mESCs were a kind gift from Dr. Ihor Lemischka (Mount Sinai School of Medicine) to I.A. They were passaged and maintained on gelatin-coated dishes or in coculture with mitomycin C (MITC)-treated mouse embryo fibroblasts (MEF) (GlobalStem) in presence of LIF (ESGRO-LIF) under previously described conditions (Schaniel et al., 2009 (link)). H9 hESCs were purchased from Wicell Institute and maintained in coculture with MITC-treated MEFs in the presence of FGF (10 ng/ml; R&D) under conditions described by the supplier. hESCs were plated on Matrigel (BD Pharmingem)-coated plates in the presence of conditioned medium from MITC-treated MEFs, FGF (10 ng/ml; R&D Systems), and ROCK inhibitor (10 μM; Stemgent) for 48 hr before compound treatment to allow hESCs to resume growth following single-cell suspension with Accutase (Innovative Cell Technologies). NTERA-2 cl.D1, 293T cells were purchased from ATCC and grown under standard tissue culture conditions. Human iPSCs, a kind gift of Drs. Mark J. Tomishima and Michel Sadelain (Papapetrou et al., 2009 (link)), were cultured under standard hESC conditions. MS436 was used at 10 μM, and MS417 was used at 250 nM concentration throughout the study. DMSO was used as a vehicle control for compounds. –(+) JQ1 enantiomers were used at 500 nM concentration for 6 hr.

Micco R.D., Fontanals-Cirera B., Low V., Ntziachristos P., Yuen S.K., Lovell C.D., Dolgalev I., Yonekubo Y., Zhang G., Rusinova E., Gerona-Navarro G., Cañamero M., Ohlmeyer M., Aifantis I., Zhou M.M., Tsirigos A, & Hernando E. (2014). Control of Embryonic Stem Cell Identity by BRD4-Dependent Transcriptional Elongation of Super-Enhancer-Associated Pluripotency Genes. Cell reports, 9(1), 234-247.

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mESC and hESC Culture Protocols

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Isolation and Culture of Human ASCs

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ASCs, originally isolated from the subcutaneous fat of healthy human, non-diabetic, non-smoking female donors, aged 18-60 years (N = 7), were purchased from Zen-Bio, Inc. (superlot #36; Research Triangle Park, NC). ASCs were grown in expansion medium consisting of DMEM/F-12 (HyClone), 10% FBS (Zen-Bio), 1% antibiotic/antimycotic (HyClone), 0.25 ng/mL transforming growth factor-β1, 5 ng/mL epidermal growth factor, and 1 ng/mL fibroblast growth factor (R&D Systems) (B. T. Estes, et al., 2008 (link)). ASCs were maintained in humidified incubators at 37°C, 5% CO₂ and passaged at 80% confluence with 0.25% trypsin-EDTA (HyClone). Experiments used ASCs at passage 4-6 (P4-6).
NHFs derived from neonatal human foreskins (a gift from Dr. Jeffrey Morgan) were expanded in high glucose DMEM (DMEM-HG, HyClone), 10% FBS (HyClone), and 1% penicillin/streptomycin (HyClone) (Youssef, et al., 2011 (link)). Experiments used NHFs at P6-9.

Beane O.S., Fonseca V.C, & Darling E.M. (2014). Adipose-Derived Stem Cells Retain their Regenerative Potential after Methotrexate Treatment. Experimental cell research, 327(2), 222-233.

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Forebrain Neurosphere Culture Protocol

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E15.5 forebrain tissue was minced and incubated with Accutase (PAA) for 15 min at 37 °C. The cell suspension was triturated with a pipet tip, passed through a 100 µm cell strainer and centrifuged at 200 × g for 3 min. The pellet was resuspended in serum-free neurosphere proliferation medium (Dulbecco’s modified Eagle’s medium/F12 medium (Gibco) supplemented with B27 supplement (Gibco), 30 mM HEPES (Gibco), 20 ng/mL epidermal growth factor (R&D), and 20 ng/mL fibroblast growth factor (R&D)). Six days after the primary tissue isolation, cells were passaged using Accutase and cultured in neurosphere proliferation medium at a density of 10⁵ cells/mL.

Filatova A., Rey L.K., Lechler M.B., Schaper J., Hempel M., Posmyk R., Szczaluba K., Santen G.W., Wieczorek D, & Nuber U.A. (2019). Mutations in SMARCB1 and in other Coffin–Siris syndrome genes lead to various brain midline defects. Nature Communications, 10, 2966.

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Generation of Colorectal Cancer Spheroids

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Colorectal cancer derived spheroid cultures were generated from tumor tissue of two CRC patients as described earlier [27 (link)]. In brief, 2008 and 2012 primary human CRC tissue or derived metastases were obtained at the University Hospital Heidelberg in accordance with the Declaration of Helsinki. Both patients signed informed consent as approved by the Ethics Review Board of the Medical Faculty, University of Heidelberg (approval number 323–2004). The tumor samples were minced and enzymatically digested with dispase (Becton, Dickinson and Company) and DNAse I (Roche). If necessary, mucus was dissolved by sputolysin (Calbiochem). The isolated cancer cells were cultivated in ultra-low attachment flasks (Corning) under serum-free conditions with addition of 10 ng/ml fibroblast growth factor (R&D Systems) and 20 ng/ml epidermal growth factor (R&D Systems) as described previously [27 (link)]. Under these conditions, multicellular spheroids formed. These spheroid cultures were authenticated and checked for contaminations with Multiplexion [28 ]. HEK-293 T cells were cultured under the same conditions as the patient-derived CRC cells. The morphology of the cells was examined via microscope (Fluorescence microscope Axiovert 200, Zeiss).

Weber S., Koschade S.E., Hoffmann C.M., Dubash T.D., Giessler K.M., Dieter S.M., Herbst F., Glimm H, & Ball C.R. (2019). The notch target gene HEYL modulates metastasis forming capacity of colorectal cancer patient-derived spheroid cells in vivo. BMC Cancer, 19, 1181.

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Mouse and Human Mesenchymal Stem Cell Isolation

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Mouse bone marrow-derived MSCs (mMSCs) were obtained from the femoral and tibial bone marrow of 7–8 weeks old Balb/c mice as previously reported [60]. The protocols were approved by the Taipei Veterans General Hospital Institutional Animal Care and Use Committee (IACUC). All studies involving animals were in accordance with appropriate guidelines. mMSCs were maintained in Low-Glucose DMEM (LG-DMEM; Invitrogen) with 10% fetal bovine serum, and 1% PSG. Commercially available human MSCs (hMSCs) (Steminent Biotherapeutics Inc, Taipei, Taiwan) were maintained in Iscove’s modified Dulbecco’s medium (IMDM; Sigma-Aldrich), supplemented with 10% fetal bovine serum, 10 ng/ml fibroblast growth factor, 10 ng/ml epidermal growth factor (R&D systems, Inc.) and 100 U Penicillin-1000 U Streptomycin-2 mM L-glutamine (1% PSG; Sigma-Aldrich).

Rimando M.G., Wu H.H., Liu Y.A., Lee C.W., Kuo S.W., Lo Y.P., Tseng K.F., Liu Y.S, & Lee O.K. (2016). Glucocorticoid receptor and Histone deacetylase 6 mediate the differential effect of dexamethasone during osteogenesis of mesenchymal stromal cells (MSCs). Scientific Reports, 6, 37371.

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Cryopreserved BM-MSCs Conditioned Media

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Cryopreserved human bone marrow-derived MSC (BM-MSC) from two healthy donors were cultured in MEM-Alpha media (Gibco) supplemented with 10% extracellular vesicle (EV)-free heat-inactivated foetal calf serum (FCS) (Gibco), 1% penicillin/streptomycin (Gibco) and 1 ng/ml fibroblast growth factor (R&D Systems, Minneapolis, MN, USA). EV-free FCS was prepared by ultracentrifugation of FCS at 100,000×g (Sorvall 100SE Ultra Centrifuge) for 18 h and subsequent collection of supernatants. Culture of human corneal endothelial cells (HCEC) was carried out in DMEM supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco). Conditioned media were prepared as described in Additional file 1: Supplementary Methods. In the case of the MSC-derived CM, this was further divided into non-manipulated CM (“MSC-CM (Whole)”) and MSC-CM from which the MSC-derived EV were depleted by ultracentrifugation for 18 h as 100,000×g (“MSC-CM (-EV)”).

Islam M.N., Griffin T.P., Sander E., Rocks S., Qazi J., Cabral J., McCaul J., McMorrow T, & Griffin M.D. (2019). Human mesenchymal stromal cells broadly modulate high glucose-induced inflammatory responses of renal proximal tubular cell monolayers. Stem Cell Research & Therapy, 10, 329.

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Adipogenic Differentiation of ASCs

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ASCs were isolated from abdomen and thigh lipoaspirate of a 56-year old, female patient with a past history of breast cancer following procedures approved by the institutional review board (IRB) of Rhode Island Hospital. Tissue was processed using previously established methods ^{12 (link)}. Prior to experiments, ASCs were expanded to third passage in medium consisting of DMEM/F-12 (Hyclone, GE Healthcare Life Sciences, Logan, UT), 10% fetal bovine serum (FBS, ZenBio, Research Triangle Park, NC), and 1% antibiotic/animycotic (Hyclone), supplemented with 5 ng/mL epidermal growth factor, 1 ng/mL fibroblast growth factor, and 0.25 ng/mL transforming growth factor-β1 (R&D Systems, Minneapolis, MN).^{13 (link)} For differentiation experiments, ASCs were exposed to either control medium containing DMEM/F-12 with 10% FBS and 1% antibiotic/antimycotic or adipogenic medium containing control medium supplemented with 0.5 μM 3-isobutyl-1-methylxanthine (IBMX), 10 μM insulin, 200 μM indomethacin, and 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO).^{37 (link)} Media were changed every two days.

Labriola N.R., Sadick J.S., Morgan J.R., Mathiowitz E, & Darling E.M. (2018). Cell Mimicking Microparticles Influence the Organization, Growth, and Mechanophenotype of Stem Cell Spheroids. Annals of biomedical engineering, 46(8), 1146-1159.

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Isolation and Differentiation of Human ASCs

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Example 1

Human ASCs were isolated from the stromal vascular fraction of donated human lipoaspirate, obtained from the abdomen and thigh of a single, 56-year old, female, breast cancer patient using method described in Estes, B. T. et al. Biotechnology and Bioengineering 2008, 99(4):986-995. Prior to use in examples, ASCs were passaged three times in expansion medium consisting of DMEM/F-12 (Hyclone, GE Healthcare Life Sciences, Logan, UT), 10% fetal bovine serum (FBS, ZenBio, Research Triangle Park, NC), 1% antibiotic/animycotic (Hyclone), supplemented with 5 ng/mL epidermal growth factor, 1 ng/mL fibroblast growth factor, and 0.25 ng/mL transforming growth factor-β1 (R&D Systems, Minneapolis, MN; Estes et al. 2008).

For differentiation examples, cells were exposed either to adipogenic or control (stromal) medium. Control medium consisted of DMEM/F-12 supplemented with 10% FBS, and 1% antibiotic/antimycotic. Adipogenic medium consisted of control medium supplemented with 0.5 μM 3-isobutyl-1-methylxanthine, 10 μM insulin, 200 μM indomethacin, and 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO) (Zheng et al. 2006). Media was refreshed every two days for all examples and ASC expansion.

US11772061B2. Methods of fabricating hyper compliant polymer particles and methods of use and compositions (2023-10-03). Brown University [US]. Inventors: Eric M. Darling [US], Nicholas R. Labriola [US], Edith Mathiowitz [US].

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Oris Cell Migration Assay for Asthma

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The Oris Cell Migration Kit was used (tebu-bio, Peterborough, United Kingdom). Triplicate repeats for vehicle control (1 μmol/L dimethyl sulfoxide), 100 nmol/L DK-PGD₂ or 100 nmol/L DK-PGD₂, and 1 μmol/L AZD6430 were added for 24 hours in 5 healthy control donors and 5 asthmatic donors. The concentrations of 500 nmol/L and 1 μmol/L DK-PGD₂ were tested in cells from 5 healthy control donors. TGF-β1 (10 ng/mL) and fibroblast growth factor (25 ng/mL; R&D Systems) were used as positive controls in cells from 5 healthy control donors and 2 asthmatic donors. Cells were fixed and labeled with Hoechst nuclear dye (Invitrogen, Carlsbad, Calif). The number of cells migrated into the migration zone was counted by a blinded observer.

Stinson S.E., Amrani Y, & Brightling C.E. (2015). D prostanoid receptor 2 (chemoattractant receptor–homologous molecule expressed on TH2 cells) protein expression in asthmatic patients and its effects on bronchial epithelial cells. The Journal of Allergy and Clinical Immunology, 135(2), 395-406.e7.

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