GL261 cells were obtained from the NCI-Frederick Cancer Research Tumor Repository (Frederick, MD). Adherent GL261 cells were cultured in DMEM (Corning) with 10% Fetal Bovine Serum (Atlanta Biologicals) and Penicillin/Streptomycin/Glutamine (Sigma-Aldrich). Cells were passaged by trypsinization and media was changed every 3–5 days. Neurosphere formation was induced by trypsinizing adherent GL261 cells and then culturing the cell suspension in the following media: serum-free DMEM with Penicillin/Streptomycin/Glutamine (Sigma-Aldrich), fibroblast growth factor (FGF-2, 20 ng/ml) (Peprotech), epidermal growth factor (EGF, 20 ng/ml) (Peprotech), and B27 (1:50) (Life Technologies). Differentiation was first noted 3–5 days after initial plating. Neurosphere culture media was replaced every 3–5 days by pelleting cells via centrifugation and re-suspending in new media. All cultures were maintained at 37.0 °C with 5.0% CO2 in 75 cm2 culture flasks (Nunc).
Fibroblast growth factor 2
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Fibroblast growth factor (FGF)-2 is a signaling protein that plays a crucial role in the regulation of cell growth, differentiation, and angiogenesis. It is a member of the FGF family of proteins and is involved in various physiological processes, including wound healing, tissue regeneration, and embryonic development.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
75 cm2 culture flask, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin glutamine (psg), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Heparin, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
U87mg, by American Type Culture Collection (1 mentions)
Human transferrin, by Merck Group (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Platelet derived growth factor, by Thermo Fisher Scientific (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Ln308, by American Type Culture Collection (1 mentions)
Ln 428, by American Type Culture Collection (1 mentions)
5 protocols using fibroblast growth factor 2
1
Culturing Glioblastoma GL261 Cells
2
Culturing U87MG and GICs Cell Lines
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U87MG was obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mmol/L L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and with (as indicated) 10% of FBS. The glioma-initiating cell (GICs) lines, ZH-161, were kindly provided by Prof. Michael Weller from the Department of Neurology at the University Hospital Zurich, Switzerland, and maintained as described [35 (link),36 (link)]. Briefly, cells were cultured in Neurobasal Medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with B-27 (20 μL/mL) and glutamax (10 μL/mL), fibroblast growth factor (FGF)-2, epidermal growth factor (EGF) (20 ng/mL each (Peprotech, Rocky Hill, PA, USA), and heparin (32 IE/mL; Sigma-Aldrich, St. Louis, MO, USA). All cells were maintained in a humidified incubator at 37 °C in 5% CO2.
3
Isolation and Culture of Oligodendrocyte Progenitor Cells
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Oligodendrocyte progenitor cells (OPC) were isolated from cortical astrocyte monolayers generated from postnatal day 1 (P1) SD pups by differential adhesion as previously described (Miron et al., 2013 ; Noble & Murray, 1984 ) . OPCs were maintained in serum‐free DMEM‐BS (adapted from Bottenstein & Sato, 1979 ) containing 0.5 mg/ml insulin in 10 mM HCL (Sigma, UK), glutamine (100 mM, Sigma), human transferrin (0.1 mg/ml, Sigma) and gentamycin (100 mg/ml, Sigma), supplemented with the growth factors; fibroblast growth factor (FGF‐2) at 10 ng/ml and platelet derived growth factor (PDGF) at 2 ng/ml (both Peprotech, UK). The isolated OPCs were plated on poly‐l ‐lysine (PLL, 13 μg/ml, Sigma) coated glass coverslips (VWR) in a 24‐well plate at a density of 5,000 cells in 50 μl drop and allowed to attach. They were maintained in DMEM‐BS containing PDGFα and FGF2 for 5 days and then switched to DMEM‐BS lacking growth factors and with or without mHeps at a concentration of 1 ng/ml. Cultures were used for proliferation, morphology, and differentiation assays.
4
Establishment and Culture of Human Glioma Cell Lines
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Human GIC (S-24, T-269, T-325, ZH-161, and ZH-305) were established from freshly resected tumors [62 (link)]. The human long-term glioma cell lines LN-18, LN-428, D247MG, LN-319, A172, LN-308, and LN-229 [63 (link)] were kindly provided by N. de Tribolet (Lausanne, Switzerland) and T98G cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). GIC were cultured as neurospheres in Neurobasal medium (NB) supplemented with 2 mM l -glutamine, 20 μg/ml B-27 supplement (Gibco, Waltham, MA), 20 ng/ml fibroblast growth factor (FGF) 2, and 20 ng/ml epidermal growth factor (EGF) (Peprotech, Rocky Hill, PA). Long-term glioma cell lines were grown as adherent monolayers in Dulbecco´s modified Eagle´s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 2 mM l -glutamine (Gibco). Cells were regularly tested for mycoplasma contamination by means of MycoAlertTM PLUS Mycoplasma Detection (Lonza, Basel, Switzerland).
5
Glioma Cell Lines and Microbubbles Protocol
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The mouse glioma cell line SMA-497 was derived from a spontaneous astrocytoma in a VM/Dk mouse [18] and provided by Dr. D. Bigner (Duke University, Durham, NC). SMA-497 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Biochrom, Minneapolis, MN) and 2 mM L-glutamine (Gibco Life Technologies, Paisley, UK). The glioma-initiating cell (GIC) line ZH-161 was established after informed consent and approval of the local ethic committees and previously characterized [19] . The cells were maintained in Neurobasal medium supplemented with 2 µl/ml B-27 without vitamin A, 2 mM L-glutamine (Gibco Life Technologies), fibroblast growth factor (FGF)-2, epidermal growth factor (EGF) (20 ng/mL each, Peprotech, Rocky Hill, PA). All cells were grown in a humidified 37 ºC incubator with 5% CO2. BG8235 microbubbles were kindly provided by Bracco Suisse SA (Geneva, Switzerland) and show similar characteristics as Bracco's BR-38 microbubbles [20] . TMZ was kindly provided by Schering-Plough (Kenilworth, NJ) and prepared in stock solutions (100 mM) in dimethylsulfoxide (DMSO). Isolated human erythrocytes were supplied by Blutspende Zürich (Zurich, Switzerland).
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