Single cells prepared from mechanical and enzymatic dissociation were seeded in 6-well ultra-low attachment plates (Corning, NY, USA) at 3000 cells per well for about 2 weeks containing serum-free DMEM/F-12 medium, B27 supplement (1 × , Invitrogen, Gaithersburg, MD, USA), 20 ng/ml human recombinant bFGF (PeproTech, Rocky Hill, NJ, USA), 20 ng/ml EGF (PeproTech), 10 ng/ml leukemia inhibitory factor (Chemicon, Tamecula, CA, USA) and 4 U/l insulin (Sigma).
Human recombinant bfgf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human recombinant bFGF is a growth factor that promotes the proliferation and differentiation of various cell types, including fibroblasts, endothelial cells, and neural cells. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human egf, by Thermo Fisher Scientific (19 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (14 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
B27 supplement, by Thermo Fisher Scientific (8 mentions)
Ultra low attachment 6 well plate, by Corning (8 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Tryple, by Thermo Fisher Scientific (5 mentions)
Accutase, by Merck Group (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (5 mentions)
Insulin, by Merck Group (4 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (4 mentions)
N2 supplement, by Thermo Fisher Scientific (4 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (4 mentions)
Leukemia inhibitory factor, by Merck Group (3 mentions)
Six well ultralow attachment plates, by Corning (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
24 well ultra low attachment plate, by Corning (3 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (3 mentions)
72 protocols using human recombinant bfgf
1
Culturing Single Cells into Spheres
2
Sphere Formation Assay for Cancer Cells
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NCI-H460/CDDP and its parental cell lines were treated DMSO, BAY-598 (200 nM), Scramble siRNA, and SMYD2 siRNA (50 nM) for 48 h, after which single cells prepared by mechanical and enzymatic dissociation were seeded in 6-well ultra-low attachment plates (Corning, NY, USA) at a density of 1,000 cells/well in serum-free DMEM/F-12 medium supplemented with B27 (1×, Invitrogen, Thermo Fisher Scientific), 20 ng/ml human recombinant bFGF (PeproTech, Rocky Hill, NJ, USA), and 20 ng/ml EGF (PeproTech) for 10–14 days. The cells were then photographed under a microscope.
3
Sphere Formation Assay for Stem Cells
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A total of 1.0 × 105 cells/well were seeded in the six-well ultra-low attachment plates (Corning, Steuben County, NY). After incubating in DMEM/F12 culture medium supplemented with 10 ng/ml human recombinant bFGF (PeproTech, Rocky Hill, NJ) and 10 ng/ml EGF (PeproTech) for 10–14 days. Sphere numbers were photographed and spheres with the diameter >50 μm were counted.
4
Spheroid Formation Assay for Cancer Cells
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T24, 5637 cells and xenografts were trypsinized by TrypLE (Life Technologies) and washed in PBS. About 1.0×104 cells per well were seeded in the 6-well ultra-low attachment plates (Corning, Steuben County, NY) in DMEM/F12 culture medium supplemented with 10 ng/ml human recombinant bFGF and 10 ng/ml EGF (PeproTech, Rocky Hill, NJ). After culturing for 9-12 days with or without TGFβ1 and/or SB-431542, spheres were photographed and spheres with the diameter greater than 50 µm were counted.
5
Differentiation of NSC-like Cells to OPCs
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NSC-like cells were dissociated into single cells using StemPro Accutase, counted and seeded at 20,000 cells/cm2 onto uncoated 6-well TC plates (Corning) or similar plates coated with human purified fibronectin (Roche) or laminin-2/merosin (Millipore). The coatings were performed by covering the well with 10 µg/ml of fibronectin or laminin-2 in PBS at 37°C for 4 h. Plates were then blocked with 0.3% Bovine Serum Albumin (BSA) in PBS for 30 min at 37°C and washed 3 times with PBS before use. The cells were cultured in NSC induction medium [22] (link) with 10 ng/ml of EGF and bFGF. At the end of 3 days, half the medium was replaced by OPC induction medium composed by Neurobasal medium supplemented with B27, 10 ng/ml of human recombinant bFGF, platelet-derived growth factor-AA (PDGF-AA) and 100 ng/ml of Sonic Hedgehog (SHH) (both from Peprotech). At the end of 3 days, the entire medium was replaced by OPC induction medium. Cells were in culture for an additional 12 days period with complete medium changes every 3–4 days.
6
Glioblastoma Stem Cell Culturing and Treatments
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We have used the same clinical materials reported in our previous papers
[24 (link), 25 (link)]. In brief, the CSC cells were retrieved from adult patients affected by GBM and undergoing craniotomy at the Institute of Neurosurgery, Catholic University-School of Medicine of Rome, Italy. Dissociated cells were cultured in the presence of human recombinant EGF (20 ng/ml; PeproTech, Rocky Hill, NJ), human recombinant bFGF (10 ng/ml; PeproTech), in DMEM/F12 (1:1) serum-free medium (Invitrogen, Carlsband, CA) containing L glutamine 2 mM, glucose 0.6%, putrescine 9.6 ug/ml, progesterone 0.025 mg/ml, sodium selenite 5.2 ng/ml, insulin 0.025 mg/ml, apo-transferrin sodium salt 0.1 mg/ml, sodium bicarbonate 3 mM, Hepes 5 mM, BSA 4 mg/ml, heparin 4 ug/ml (all purchased by Sigma-Aldrich). Floating neurospheres were dissociated with Accutase at 37°C (Merck-Millipore). In some cases, neurospheres were passaged up to passage P60 and the experiments were performed between P14 and P60. Cell starvation was planned for 2 days in Stem Medium w/o EGF and bFGF. Subsequently, PDGF-AA was added (40 ng/ml, Peprotech) for different time points (5′, 10′, 30′, 120′ and 24 hours). Cells treatments were performed with GSI-X (also named L-685,458, Calbiochem), AG1478 (Calbiochem) and Crenolanib (CP-8685596, Selleckchem).
[24 (link), 25 (link)]. In brief, the CSC cells were retrieved from adult patients affected by GBM and undergoing craniotomy at the Institute of Neurosurgery, Catholic University-School of Medicine of Rome, Italy. Dissociated cells were cultured in the presence of human recombinant EGF (20 ng/ml; PeproTech, Rocky Hill, NJ), human recombinant bFGF (10 ng/ml; PeproTech), in DMEM/F12 (1:1) serum-free medium (Invitrogen, Carlsband, CA) containing L glutamine 2 mM, glucose 0.6%, putrescine 9.6 ug/ml, progesterone 0.025 mg/ml, sodium selenite 5.2 ng/ml, insulin 0.025 mg/ml, apo-transferrin sodium salt 0.1 mg/ml, sodium bicarbonate 3 mM, Hepes 5 mM, BSA 4 mg/ml, heparin 4 ug/ml (all purchased by Sigma-Aldrich). Floating neurospheres were dissociated with Accutase at 37°C (Merck-Millipore). In some cases, neurospheres were passaged up to passage P60 and the experiments were performed between P14 and P60. Cell starvation was planned for 2 days in Stem Medium w/o EGF and bFGF. Subsequently, PDGF-AA was added (40 ng/ml, Peprotech) for different time points (5′, 10′, 30′, 120′ and 24 hours). Cells treatments were performed with GSI-X (also named L-685,458, Calbiochem), AG1478 (Calbiochem) and Crenolanib (CP-8685596, Selleckchem).
7
Sphere-Formation Assay for Cell Self-Renewal
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To evaluate the self-renewal activity of each cell line, a sphere-formation assay was performed using the protocol described in a previous report with a slight modification62 (link). Cells were seeded at 2000 cells/mL in an ultra-low attachment 24-well plate (Corning). These cells were cultured in serum-free DMEM/F12 (Nacalai Tesque) supplemented with bovine serum albumin (Nacalai Tesque), 5 mM HEPES (Nacalai Tesque), 100 U/mL penicillin (Nacalai Tesque), 100 µg/mL streptomycin (Nacalai Tesque), 20 ng/mL human recombinant EGF (Peprotech, NJ), 10 ng/mL human recombinant bFGF (Peprotech), and 1% B27 supplement (Invitrogen, CA). EGF, bFGF, and B27 supplement were used to stabilise the formation and maintenance of sphere. DMEM/F12 medium supplemented with 2 ng/mL EGF and 1 ng/mL bFGF was added to the culture every other day for 10 days. Spheres were counted under a light microscope at 40 × magnification. Each experiment was performed in triplicate and repeated at least thrice.
8
Isolation of GBM Cancer Stem Cells
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Procedures for collection of adult human GBM CSCs were approved by the Ethical Committee of the Catholic University of Rome, as previously reported [5 (link)]. The ethical principles of the Declaration of Helsinki were strictly followed.
CSC cells were retrieved from three adult patients affected with GBM and undergoing craniotomy at the Institute of Neurosurgery, Catholic University School of Medicine of Rome, Italy. Cells grow spontaneously in suspension as neurospheres in the presence of human recombinant EGF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) and human recombinant bFGF (10 ng/mL; PeproTech) in serum-free medium DMEM/F12 (1:1) (Invitrogen, Thermo Fisher), as previously described. Mid-sized neurospheres were enzymatically dissociated using Accutase (Merck Millipore, Darmstadt, Germany) for 1–2 min at 37 °C and replated as single cells for healthy cell proliferation. In order to have good preparations in terms of abundance and integrity of sEVs, CSCs from each patient were plated on matrigel-pre-coated 175 cm2 flasks to achieve 80–90 percent confluence by 48 h. Before moving on to the sEV purification process, the supernatants were pooled together.
CSC cells were retrieved from three adult patients affected with GBM and undergoing craniotomy at the Institute of Neurosurgery, Catholic University School of Medicine of Rome, Italy. Cells grow spontaneously in suspension as neurospheres in the presence of human recombinant EGF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) and human recombinant bFGF (10 ng/mL; PeproTech) in serum-free medium DMEM/F12 (1:1) (Invitrogen, Thermo Fisher), as previously described. Mid-sized neurospheres were enzymatically dissociated using Accutase (Merck Millipore, Darmstadt, Germany) for 1–2 min at 37 °C and replated as single cells for healthy cell proliferation. In order to have good preparations in terms of abundance and integrity of sEVs, CSCs from each patient were plated on matrigel-pre-coated 175 cm2 flasks to achieve 80–90 percent confluence by 48 h. Before moving on to the sEV purification process, the supernatants were pooled together.
9
Differentiation of Human Embryonic Stem Cells
10
Spheroid Formation Assay for Cancer Cells
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T24 and 5637 cells were trypsinized by TrypLE (Life Technologies Corp., Grand Island, NY, USA) and washed by PBS. 1.0×105 cells per well were seeded in the 6-well ultra-low attachment plates (Corning, Steuben County, NY, USA) in DMEM/F-12 culture medium supplemented with 10 ng/ml human recombinant bFGF and 10 ng/ml EGF (PeproTech, Rocky Hill, NJ, USA). After culture for 14 days with or without honokiol, spheres were photographed. Spheres with the diameter greater than 50 μm were counted.
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