The biphasic calcium phosphate (BCP) granule (Osteon GBC0305,) was obtained from Dentium Corporation, Suwon, Korea, and FGF-2 was obtained from the Genoss Corporation, Suwon, Korea, respectively. BCP is a 70% hydroxyapatite (HAp) scaffold coated with 30% β-tricalcium phosphate (β-TCP) with a particle size of 0.3–0.5 μm. As previously reported [22 (link)], the procedure for preparing FGF-2 coated BCP (see Figure 1 ) includes the grafting of 3-aminopropyl triethoxysilane (APTES, Sigma, MO, USA) onto the BCP surface and the replacement of the terminal amine by a maleimide functional group that reacts with FGF-2 via a heterobifunctional cross-linker (N-Succinimidyl-3-maleimidopropionate: SMP, Sigma, MO, USA). Briefly, 1.0 g of BCP powder was silanized by 10 mM APTES dissolved in hexane (Sigma, MO, USA) for 2 h. The silanized BCP powder was substituted for maleimide groups by 2 mM SMP dissolved in anhydrous dimethylformamide (DMF, Sigma, MO, USA) for 2 h. Then, FGF-2 dissolved in anhydrous DMF was immobilized on BCP by stirring for 2 h. All experimental procedures were performed at 25°C to avoid the risk of FGF-2 denaturation.
Fibroblast growth factor 2 (fgf2)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Macao, Australia
FGF2 is a recombinant human fibroblast growth factor 2 protein. It is a potent mitogen that stimulates the proliferation of a variety of cell types, including fibroblasts, myoblasts, osteoblasts, and endothelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Merck Group (58 mentions)
Dmem f12, by Thermo Fisher Scientific (24 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (19 mentions)
Heparin, by Merck Group (19 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
N2 supplement, by Thermo Fisher Scientific (11 mentions)
Glutamax, by Thermo Fisher Scientific (11 mentions)
B27 supplement, by Thermo Fisher Scientific (10 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (10 mentions)
L glutamine, by Thermo Fisher Scientific (9 mentions)
Bovine serum albumin (bsa), by Merck Group (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Insulin, by Merck Group (8 mentions)
β mercaptoethanol, by Merck Group (8 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (7 mentions)
Matrigel, by BD (7 mentions)
Laminin, by Merck Group (6 mentions)
Accutase, by Merck Group (6 mentions)
215 protocols using fibroblast growth factor 2 (fgf2)
1
FGF-2 Coated Biphasic Calcium Phosphate Granules
2
Murine Neural Stem Cell Derivation
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Primary neural stem/progenitor cells (NSCs) were derived from male mice of a mixed MF1-derived background. Three batches were used in this study: batch X6 (from adult subventricular zone tissue, age 8 weeks), batches X8 and X9 (from P3 postnatal cortical grey matter tissue).
Tissue was dissociated with the ‘Neural Tissue Dissociation Kit (P)’ (Miltenyi, 130-092-628) and cell preparations were initially expanded for 1–2 passages as neurospheres in basal medium ((DMEM/F-12 + Glutamax (Invitrogen 31331-093) + 1× N2 supplement (R&D Systems, AR009) + 1× penicillin–streptomycin (ThermoFischer Scientific, 15140)) supplemented with 20 ng/ml EGF (Preprotech, 315-09-100) + 20 ng/ml FGF2 (Peprotech, 450-33) + 5 μg/mL Heparin (Sigma, H3393-50KU).
Subsequently cells were further expanded as adherent cultures in basal medium supplemented with EGF + FGF2 + Heparin (as above), + 2 μg/ml Laminin (Sigma, L2020) (EGF/FGF2-medium), on plates pre-coated for > 1 h with EGF/FGF2 medium. For passaging, cells were detached with Accutase (Sigma, A6964). Cells were maintained at 37 °C, 5% CO2 and used for experiments at total passage 7–25.
Tissue was dissociated with the ‘Neural Tissue Dissociation Kit (P)’ (Miltenyi, 130-092-628) and cell preparations were initially expanded for 1–2 passages as neurospheres in basal medium ((DMEM/F-12 + Glutamax (Invitrogen 31331-093) + 1× N2 supplement (R&D Systems, AR009) + 1× penicillin–streptomycin (ThermoFischer Scientific, 15140)) supplemented with 20 ng/ml EGF (Preprotech, 315-09-100) + 20 ng/ml FGF2 (Peprotech, 450-33) + 5 μg/mL Heparin (Sigma, H3393-50KU).
Subsequently cells were further expanded as adherent cultures in basal medium supplemented with EGF + FGF2 + Heparin (as above), + 2 μg/ml Laminin (Sigma, L2020) (EGF/FGF2-medium), on plates pre-coated for > 1 h with EGF/FGF2 medium. For passaging, cells were detached with Accutase (Sigma, A6964). Cells were maintained at 37 °C, 5% CO2 and used for experiments at total passage 7–25.
3
Live Cell Imaging of Random Cell Migration
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For live cell imaging of random cell migration cells were seeded at 1 × 105/well in flat-bottomed 96 well plates (Nunc) with or without inhibitor treatment in full medium, serum-free Heparan sulphate proteoglycan (HSPG) (Sigma) only supplemented medium or serum-free HSPG/FGF2 supplemented medium. FGF2 (Sigma) was added at 10 ng/ml and HSPG (Sigma) at 10 μg/ml. Cell migration was imaged with the IncuCyte ZOOM system (Essen BioScience) over 72 h at hourly intervals at x10 magnification. Avi formatted movies were analyzed using ImageJ1 .
For SF188 and KNS42, at least 20 cells per condition/recorded field were identified and random migratory patterns recorded by tracking individual nuclei with MTrackJ (https://imagescience.org/meijering/software/mtrackj/ ) to determine velocity and directionality (16 ). For pLGG, up to 10 cells/condition/field were identified because of low cell proliferation rates. Rose plots were generated using Ibidi Chemotaxis PlugIn.
For SF188 and KNS42, at least 20 cells per condition/recorded field were identified and random migratory patterns recorded by tracking individual nuclei with MTrackJ (
4
Hypothalamic Neural Progenitor Cell Culture
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The hypothalamus was dissected from newborns, and three hypothalami from the same litter were pooled and placed in DMEM/F12 medium, trypsinized, and cultured as previously reported [38 (link)]. Briefly, NPCs were cultured in complete medium [NeurobasalTM Medium containing 1% anti-anti (Invitrogen, Waltham, MA, USA), 2% B27 (GIBCO, Cat#17504-044), 20 ng/mL FGF2 (Sigma, St. Louis, MI, USA), 20 ng/mL EGF (Sigma), 1 μg/mL heparin (Lilly, Indianapolis, IN, USA), and 2.5 μg/mL L-glutamine (Invitrogen)] or differentiation medium (cultured in absence of FGF2, EGF and heparin) and seeded in culture dishes pre-coated with 0.01% poly-L-lysine (Sigma).
5
Isolation and Differentiation of Neural Progenitor Cells
6
Rat Cortex Neurosphere Differentiation
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NSCs were isolated from E14 Sprague-Dawley rat cortex (Orient Bio, Seongnam, Republic of Korea) and expanded as a neurosphere in Dulbecco’s modified Eagle medium/F12 supplemented with 1% (v/v) antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA, USA), 2% (v/v) B27 (Thermo Fisher Scientific), and 20 ng/mL each of EGF and FGF2 (Merck Millipore). After 6 days, the cells were dissociated, plated onto 0.01% poly-d -lysine (Sigma-Aldrich, St. Louis, MO, USA) and 10 μg/mL laminin (Thermo Fisher Scientific) coated plates and expanded for 1 day. The final differentiation step involved removing EGF/FGF2 and treating with 0.1% DMSO (Sigma-Aldrich), 20 μM PD98059 (Merck Millipore), or 20 μM the synthesized compounds (Table 1 ) after 1 h, 3 days, or 4 days. Animal experiments were performed following Chung-Ang University and NIH standards of animal care.
7
Rat BMSC Isolation and Transfection
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Rat BMSCs were isolated from the femurs and tibias of male Wistar rats (90–100 g) as described earlier with modification [24 (link)]. Rats were killed by intravenous injection of ketamine/xylazine, and all work was performed according to the Institutional Animal Care and Use Committee guidelines. This experimental protocol was reviewed and approved by the Medical Ethics Committee of Shandong University School of Medicine. Briefly, the cells were seeded in Dulbecco's modified Eagle's medium low glucose (Gibco, USA) supplemented with 15% fetal bovine serum and fibroblast growth factor 2 (FGF-2) (Beifuji, China), 10 ng/mL, at 37°C in humidified air with 5% CO2. Rat BMSCs were phenotypically characterized as described [25 (link)]. Cells were transfected with pCMV6 empty vector/pCMV6-CD163 in DMEM with FGF-2 (10 ng/mL), rVEGF (10 ng/mL) (Sigma, St. Louis, MO), and 2% serum and incubated at 37°C for 24 h. Then, the medium is replaced by basal DMEM with FGF-2 (10 ng/mL) and rVEGF (10 ng/mL) incubated at 37°C for another 24 h.
8
Oxytocin and FGF2 Effects on Ultrasonic Vocalizations
9
Molecular Inhibitors in Cell Culture
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Chemicals used in this study were as follows: gefitinib (JS Research Chemicals Trading e. Kfm., Schleswig Holstein, Germany), erlotinib (Cayman Chemical Company, Ann Arbor, MI, USA), PD173074 (Merck Millipore, Billerica, MA, USA), MG132 (Cell signaling technology, Danvers, MA, USA) and FGF2 (ReproCELL Incorporated, Yokohama, Japan). The agents except for FGF2 were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA). For neutralization of extracellular FGF2 in culture medium, anti-FGF2 antibody (#05–117, clone bFM-1; Merck Millipore) was used.
10
3D Organoid Culture of Mouse Mammary Epithelial Cells
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Three-dimensional organotypic culture was performed in growth factor-reduced BM from Engelbreth–Holm–Swarm mouse sarcoma (Matrigel, Becton Dickinson), which was prepared according to the manufacturer's instructions. Isolated primary MMECs grown on low adhesion plates were trypsinized with 0.05% Trypsin-EDTA for 5-10 min, centrifuged and suspended with liquid Matrigel, and plated onto 8-well chamber slides at ∼1500 cells/well. Organoids were grown in DMEM/F12 supplemented with ITS medium supplement (Sigma-Aldrich), penicillin and streptomycin and 2.5 nM FGF-2 (Sigma-Aldrich). Wnt3a and Wnt4 (both R&D Biosystems) were used in 100 ng/ml, Rspo1 (R&D Biosystems) in 500 ng/ml, and porcupine inhibitor IWP-2 (Sigma-Aldrich) in 2 µM, and were added on the starting day of the cultures. Medium was refreshed every 3-4 days. For lactogenic differentiation, organoids were first grown for 6 days in the DMEM/F12 medium supplemented with 2.5 nM FGF-2, ITS and penicillin and streptomycin, and thereafter, 6 days in the same culture medium additionally supplemented with 1 μg/ml mouse prolactin (Sigma-Aldrich) and 1 μg/ml hydrocortisone (Sigma-Aldrich).
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