mGSC-F3-T3-shTP53 and mGSC-HRAS-12V-shTP53 were isolated from the brains of mice that had received injection of lentivirus containing a bi-cistronic expression cassette including F3-T3 or HRAS-12V and TP53-shRNA into the dentate gyrus as described4 (link),7 (link). 2–4 months after intracranial injection mice showing neurological symptoms were sacrificed, brain tumors were identified macroscopically, dissected and cultured in DMEM:F12 containing 1× N2 and B27 supplements (Invitrogen) and human recombinant FGF-2 and EGF (20 ng ml−1 each; Peprotech). Studies were approved by the IACUC at Columbia University (#AAAL7600).
Human recombinant fgf 2 and egf
Manufactured by Thermo Fisher Scientific
Human recombinant FGF-2 and EGF are growth factors commonly used in cell culture applications. FGF-2 (also known as basic fibroblast growth factor) and EGF (epidermal growth factor) are proteins that stimulate cell growth, proliferation, and differentiation. These recombinant proteins are produced using genetic engineering techniques.
Automatically generated - may contain errors
Lab products found in correlation
N2 and b27 supplements, by Thermo Fisher Scientific (4 mentions)
Mouse laminin, by Merck Group (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Primaria dishes, by BD (2 mentions)
Cell strainer, by BD (1 mentions)
Poly l ornithine solution, by Merck Group (1 mentions)
Accutase, by Merck Group (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
4 protocols using human recombinant fgf 2 and egf
1
Isolation of Mouse Glioma Stem Cells
2
Dissociation and Culture of Tumor Cells
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Brains with MB were dissected out and transferred into PBS on ice. Blood and fat tissues were removed from the tumor mass under a dissecting microscope and the tumor tissue pieces were washed again with PBS. Tumor tissues were minced with a scalpel, passed through syringes with 18- and 22-gauge needles, and then incubated in a 1 : 1 mixture of Accutase (Sigma) and TrypLE (Invitrogen) for 15 min at 37 °C with occasionally vortex. Undigested tissues were removed by filtering with 40 μm cell strainer (Falcon, 352340). Dissociated cells were washed twice with Dulbecco’s modified Eagle medium (DMEM)/F12 medium followed by centrifugation at 2000 r.p.m. for 5 min. Cells were then resuspended and plated onto uncoated dishes in Neurobasal and DMEM/F12 media (1 : 1 mix) containing N2 and B27 supplements (Invitrogen), and human recombinant FGF2 and EGF (20 ng/ml, PEPROTECH). Five to 7 days later, cell spheres were dissociated in Accutase (Sigma-Aldrich) and plated onto Primaria dishes (BD Biosciences) coated with Poly-l -ornithine solution (Sigma-Aldrich) and mouse laminin (Sigma-Aldrich) to allow adherent growth.
3
Isolation of Mouse Glioma Stem Cells
4
Establishing Patient-Derived Tumor Spheres
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Tumor tissue collected after surgery was minced with a scalpel, passed through syringes with 18- and 22-gauge needles, and then incubated in a 1:1 mixture of Accutase (eBioscience, San Diego, CA, USA) and TrypLE (Invitrogen) for 10 min at 37 °C. The dissociated cells were washed twice with DMEM medium followed by centrifugation at 500 × g for 8 min before being plated onto uncoated dishes in Neurobasal media and DMEM media (1:1 mix) containing N2 and B27 supplements (Invitrogen) and human recombinant FGF2 and EGF (10 ng/ml, PEPROTECH). Five to 7 days later, the spheres were plated onto Primaria dishes (BD Biosciences) coated with mouse laminin (Sigma-Aldrich) to allow adherent growth as described previously73 (link). Cells were maintained and passaged as adherent cultures.
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