Recombinant human fgfb

Skeletal muscle progenitors were isolated from hindlimbs muscles of 8–10 weeks old mice as described in^{92 (link)}. Briefly, muscles were finely minced and digested with Collagenase A for 1 h at 37 °C. Cells were centrifuged at 500 g for 10 min and supernatant was removed. Cell pellet was resuspended in full myoblast (MB) proliferation medium containing 20% FCS, 10% HS, 2.5 ng/ml recombinant human FGFb (Gibco), 0.5% chicken embryo extract (ZellBio). The pellet was placed on Matrigel (Corning) coated plates (working concentration of 0.9 mg/ml) where myoblast migration from the minced myofibers was observed on day 3 of culture. At that point, cells were trypsinized and plated for 1 h on type I rat tail collagen (working concentration 0.1 mg/ml) (Corning) coated plates for removal of fibroblastic and non-myogenic cells. Next, medium containing non-attached cells was collected and placed on Matrigel coated plates for expansion of the myoblast culture.

We have developed a characterised GBM patient-derived cell line resource (Q-Cell) [10 (link),11 (link),12 (link),13 (link)], in which lines are maintained as glioma neural stem cell (GNS) cultures [14 (link)] or as 3D neurosphere cultures [15 (link)] using StemPro NSC SFM (Invitrogen, Carlsbad, CA, USA) or KnockOut™ DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s guidelines. Characterisation data is freely available from https://www.qimrberghofer.edu.au/q-cell/. All tissues were collected following ethical approval from the Royal Brisbane Women’s Hospital and QIMR Berghofer Human Research Ethics Committees. Ethical approval number: P3420, HREC/17/QRBW/577 Novel Therapies for Brain Cancer. In order to maintain pluripotency, KnockOut™ DMEM (Gibco) media were supplemented with GlutaMAX™ Supplement (Gibco), StemPro™ Neural Supplement (Gibco), Recombinant Human EGF (Gibco), Recombinant Human FGFb (Gibco), and Penicillin/Streptomycin (Gibco). Cells were cultured on flasks coated with Basement Membrane Matrigel® Matrix (Corning, New York, NY, USA). Passaging the cells was done by detaching the cells from the flask surface using Accutase® solution (Sigma-Aldrich, St. Louis, MO, USA). Glioma cells were cultured as glioma neural stem (GNS) cultures as outlined in detail in [14 (link)] or as tumourspheres using StemPro^® NSC SFM.

Glioblastoma primary cell lines and culturing techniques were described previously [20 (link),21 (link)] and followed the basic protocol outlined by Pollard and colleagues [22 (link)]. Briefly, cell culture media included KnockOut™ DMEM/F-12, GlutaMAX™ Supplement, StemPro™ Neural Supplement, Recombinant Human EGF, Recombinant Human FGFb, and Penicillin/Streptomycin (Thermo Fisher Scientific, Scoresby, VIC, Australia). Cells were cultured on flasks coated with Basement Membrane Matrigel^® Matrix. Cell passaging was done by detaching the cells from the flask surface using Accutase^® solution (Sigma Aldrich, North Ryde, NSW, Australia, cat. H9268).