Mammalian cells were cultured using standard protocols. Cells were grown in Hyclone DMEM with high glucose (GE Healthcare Life Sciences) supplemented with L-glutamine (Wisent Bioproducts), penicillin-streptomycin (Wisent Bioproducts), and 10% bovine calf serum (GE Healthcare Life Sciences) at 37°C in 5% CO2. The previously characterized glioblastoma patient surgical specimen–derived BTICs, BT048 and BT025, were maintained as described (Kelly et al., 2009 (link); Verginelli et al., 2013 (link)). BTICs were cultured in serum-free medium (NeuroCult proliferation medium; Stemcell Technologies) supplemented with 2 µg/ml heparin sulfate (Stemcell Technologies) or serum-free medium supplemented with 20 ng/ml human recombinant epidermal growth factor (PeproTech), 20 ng/ml recombinant human fibroblast growth factor (PeproTech), and 2 µg/ml heparin sulfate. The BTICs give rise to neurospheres that are evident as early as 7 d after plating. Neurospheres were grown until they reached a size adequate for passaging (∼100–200 µm).
Heparin sulfate
Manufactured by STEMCELL
Heparin sulfate is a highly sulfated glycosaminoglycan that is naturally found in the extracellular matrix of various tissues. It is commonly used as a key component in cell culture media to support the growth and maintenance of diverse cell types.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Wisent (2 mentions)
Bovine calf serum (bcs), by GE Healthcare (2 mentions)
Heparin sulfate, by Thermo Fisher Scientific (2 mentions)
Neurocult proliferation medium, by STEMCELL (2 mentions)
L glutamine, by Wisent (2 mentions)
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions)
Recombinant human fibroblast growth factor, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
L glutamine, by Corning (1 mentions)
Human bmp4, by R&D Systems (1 mentions)
Mem neaa, by Merck Group (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Pfrs2 y196, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Tgf β, by Cell Signaling Technology (1 mentions)
Anti fgfr1, by Cell Signaling Technology (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
4 protocols using heparin sulfate
1
Culturing Glioblastoma Stem Cells
2
Trophoblast Differentiation from hPSCs
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Trophoblast differentiation from hPSCs using BMP4 was performed as previously described (Horii et al., 2016 (link)). Prior to differentiation, primed or naïve hPSCs were adapted to culture in StemPro (Thermo Fisher, A1000701) medium with 12 ng/mL recombinant basic FGF (bFGF) (Thermo Fisher, PHG0261) on Geltrex coated plates. Naïve and capacitated hPSCs were directly subjected to trophoblast differentiation conditions. Briefly, 2.0 × 104 to 1.0 × 105 primed, re-primed, naïve, or capacitated hPSCs were seeded on Geltrex coated 24-well plates in EMIM medium [DMEM/F12 supplemented with 1X MEM NEAA, 2% BSA (Sigma-Aldrich, A9418), 2 mM L-Glutamine (Corning, 25–005 CI), 1% ITS (Gibco, 41400), and 100 ng/mL heparin sulfate (Stemcell Technologies, 07980] for 2 days. They were cultured in EMIM supplemented with 10 ng/mL human BMP4 (R and D Systems, 314 BP) for four more days, at which point they were designated as CTBs. For terminal EVT and STB differentiation, the CTBs were further cultured in FCM [DMEM/F12 supplemented with 1X GlutaMAX, and 1% non-essential amino acid, 0.1 mM β-mercaptoethanol; conditioned on irradiated MEFs for 24 hr] supplemented with 10 ng/mL human BMP4 for 6 days. Media were changed every 1–2 days. Cells were cultured in 5% CO2 and 20% O2.
3
FGF2 and FGFR1 Signaling Pathway Assay
4
Culturing Glioblastoma Brain Tumor-Initiating Cells
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Mammalian cells were cultured using standard protocols. Cells were grown in HyClone Dulbecco's Modified Eagle Medium with high glucose (GE Healthcare Life Sciences) supplemented with L-Glutamine (WISENT Inc), penicillinstreptomycin (WISENT Inc) and 10% bovine calf serum (GE Healthcare Life Sciences) at 37˚C in 5% CO2. The previously characterized glioblastoma patient surgical specimen-derived BTICs, BT048 and BT025 were maintained as described (Kelly et al., 2009; (link)Verginelli et al., 2013) (link). BTICs were cultured in serum-free medium (SFM) (NeuroCult proliferation medium; STEMCELL Technologies) supplemented with 2 µg/ml heparin sulfate (STEMCELL Technologies) or SFM supplemented with 20 ng/ml human recombinant epidermal growth factor (hEGF; PeproTech) and 20 ng/ml recombinant human fibroblast growth factor (hFGF; PeproTech) and 2 µg/ml heparin sulfate. The BTICs give rise to neurospheres that were evident as early as 7 days after plating. Neurospheres were grown until they reached a size (~100-200 μm) adequate for passaging.
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