Fgf8b

Manufactured by R&D Systems

Sourced in United States

FGF8b is a recombinant human Fibroblast Growth Factor 8 isoform b. It is a member of the fibroblast growth factor family and is involved in various biological processes.

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Lab products found in correlation

Brain derived neurotrophic factor (bdnf), by R&D Systems (5 mentions) Ascorbic acid, by Merck Group (4 mentions) Noggin, by R&D Systems (2 mentions) Fibroblast growth factor 2 (fgf2), by R&D Systems (2 mentions) Laminin, by Merck Group (2 mentions) Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions) Mem non essential amino acids solution, by Thermo Fisher Scientific (2 mentions) B27 supplement, by Thermo Fisher Scientific (2 mentions) Purmorphamine, by Merck Group (2 mentions) 2 mercaptoethanol, by Fujifilm (1 mentions) Knockout serum replacement ksr, by Thermo Fisher Scientific (1 mentions) Cyclopamine, by Enzo Life Sciences (1 mentions) Trypsin edta, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Sb431542, by Merck Group (1 mentions) Heparin coated acrylic beads, by Merck Group (1 mentions) Sonic hedgehog, by R&D Systems (1 mentions) Dorsomorphin dihydrochloride, by Bio-Techne (1 mentions) Valproic acid, by Merck Group (1 mentions) Putrescine, by Merck Group (1 mentions)

14 protocols using fgf8b

Differentiation of mESCs into Dopaminergic Neurons

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R1 mESCs (Nagy’s lab, MSH, Toronto, Canada) were cultured and differentiated on PA6 stromal feeders (RCB1127, Riken BRC Cell Bank, Japan)43 (link). Specifically, mESCs were plated at low density (100 cells/cm²) on a confluent layer of PA6 cells and were grown in Serum Replacement Medium with Noggin (300 ng/ml; R&D Systems) as previously described44 (link). At day 5, 200 ng/ml Shh (R&D Systems) and 25 ng/mL Fgf8b (R&D Systems) were added to the medium. At day 8, the medium was switched to N2 medium containing Shh, Fgf8b, and Fgf2 (10 ng/ml, R&D Systems). At day 10 of differentiation the cells were pulsed with EdU (10 μM, Life Technologies). From day 11 of differentiation, Shh, Fgf8b and Fgf2 were removed from the N2 medium and replaced by BDNF (20 ng/ml, R&D Systems), GDNF (20 ng/ml, R&D Systems), and ascorbic acid (0.2 mM). Between day 8–15, cells were treated daily with either dopamine (10 μM), haloperidol (1 μM), quinpirole (10 μM), sulpiride (10 μM) or media only. At day 15, cells were fixed in 4% PFA and processed for immunocytochemistry as described below.

Hedlund E., Belnoue L., Theofilopoulos S., Salto C., Bye C., Parish C., Deng Q., Kadkhodaei B., Ericson J., Arenas E., Perlmann T, & Simon A. (2016). Dopamine Receptor Antagonists Enhance Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors. Scientific Reports, 6, 26448.

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Differentiation of Mouse ESCs into Cortical Neurons

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Bcl11b-IRES-EGFP knock-in mouse ESCs were maintained and cultured as previously reported (Watanabe et al., 2005 (link)). The differentiation medium was constituted of G-MEM supplemented with 10% Knockout Serum Replacement (KSR; Invitrogen), 2 mM L-glutamine (Thermo Fisher Scientific), 1 mM Sodium pyruvate solution (SIGMA), 0.1 mM MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific), 0.1 mM 2-Mercaptoethanol (Wako, Japan), 10 μM SB431542 (Merck), and 20 nM Wnt-C59 (Cellagen Technology) (Eiraku et al., 2008 (link); Motono et al., 2016 (link)). For the SFEBq culture, ESCs were dissociated into single cells in 0.05% trypsin-EDTA (Invitrogen) and quickly reaggregated in the differentiation medium (4000 cells/150 μl/well) using Prime Surface 96U plates (Sumilon). To induce mouse ESC-derived cortical neurons, day 6 cell aggregates were transferred to a 10 cm bacterial-grade dish in N2 medium (DMEM/F12 supplemented with N2, B27, 0.1 mM 2-ME, and 2 mM glutamine) supplemented with 50 ng/ml FGF8b (R&D systems) and 5 μM cyclopamine (Enzo life sciences) for dorso-anteriorization of the telencephalon.

Sunohara T., Morizane A., Matsuura S., Miyamoto S., Saito H, & Takahashi J. (2019). MicroRNA-Based Separation of Cortico-Fugal Projection Neuron-Like Cells Derived From Embryonic Stem Cells. Frontiers in Neuroscience, 13, 1141.

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Protein-coated Beads for Chick Embryo

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Heparin-coated acrylic beads (Sigma Aldrich, MO, USA; H5263) were added to 0.1% BSA/PBS (FUJIFILM Wako, Japan; 013–15104) solution or 100 ng/μL or 500 ng/μL FGF8b (R&D systems, MN, USA; 423-F8-025)/0.1% BSA solution and allowed to adsorb proteins for 1 h at room temperature. The beads were then implanted ventrally into the chick embryos and cultured on the culture gel described above.

Saito S., Kanazawa U., Tatsumi A., Iida A., Takemoto T, & Suzuki T. (2024). Functional analysis of a first hindlimb positioning enhancer via Gdf11 expression. Frontiers in Cell and Developmental Biology, 12, 1302141.

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Efficient Stem Cell Culture Protocols

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Gelatine, putrescine, sodium selenite, progesterone, apotransferrin, glucose, insulin, ascorbic acid, valproic acid and ICRT3 were obtained from Sigma (Steinheim, Germany). Accutase was from PAA (Pasching, Austria). FGF-2 (basic fibroblast growth factor), FGF-8b, Sonic hedgehog and noggin and were obtained from R&D Systems (Minneapolis, MN, USA). Y-27632, SB-43154, CHIR99021 and dorsomorphin dihydrochloride were from Tocris Bioscience (Bristol, UK). MatrigelTM was from BD Biosciences (Massachusetts, USA). All cell culture reagents were from Gibco/Invitrogen (Darmstadt, Germany) unless otherwise specified.

Meisig J., Dreser N., Kapitza M., Henry M., Rotshteyn T., Rahnenführer J., Hengstler J.G., Sachinidis A., Waldmann T., Leist M, & Blüthgen N. (2020). Kinetic modeling of stem cell transcriptome dynamics to identify regulatory modules of normal and disturbed neuroectodermal differentiation. Nucleic Acids Research, 48(22), 12577-12592.

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Neural Induction of Pluripotent Stem Cells

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mESC lines (EB5 (ref. 38 (link)); passages 35–45, LMX1A::GFP KI ESCs; passages 11-21, G4-2 (ref. 38 (link)); passages 20–30) and iPSC line (440A-3, a kind gift from Dr Okita, Kyoto University Center for iPS Cell Research and Application, Kyoto, Japan; passages 15–25) were maintained on mitotically inactivated mouse embryo fibroblast feeder layer in knockout DMEM medium supplemented with 1% penicillin/streptomycin (P/S; Gibco), 20% fetal bovine serum (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (2-ME; Wako), 2 mM L-glutamine (L-Glu; Sigma-Aldrich), 2,000 U ml⁻¹ LIF (Merck Millipore) and 1 × Nucleosides (Merck Millipore). We changed the medium every day.
For neural induction, mESCs and miPSCs were replated in low cell adhesion 96-well plates (Lipidure-Coat Plate A-96U; NOF Corporation) at a density of 9,000 cells per well in a differentiation medium containing Glasgow minimum essential medium (GMEM) (Gibco) supplemented with 5% KSR, 0.1 mM MEM non-essential amino acids solution (Gibco), 2-ME, 1 mM sodium Pyruvate solution (Pyruvate; Sigma-Aldrich) and 2 mM L-Glu. Moreover, we added both 100 ng ml⁻¹ FGF8b (R&D) and SHH (R&D) to induce midbrain and FP cells, respectively, from day 1 to day 6. On day 7, we added 200 nM Ascorbic acid (AA; Nacalai), 20 ng ml⁻¹ brain-derived neurotrophic factor (BDNF) (R&D), 1 × N-2 supplement (Gibco) and removed 5% KSR. We changed the medium every 2 days.

Samata B., Doi D., Nishimura K., Kikuchi T., Watanabe A., Sakamoto Y., Kakuta J., Ono Y, & Takahashi J. (2016). Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1. Nature Communications, 7, 13097.

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Dopaminergic Neuron Induction Protocol

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To induce DA neurons, the medium was replaced with NM containing 20 ng/mL bFGF and 20 ng/mL EGF at 3 days after transduction for 3 days. Next, 200 ng/mL Shh and 100 ng/mL FGF8b (R&D Systems) were added to the medium and the cells were cultured for an additional 9 days. The medium was changed with NM supplemented with only both Shh (200 ng/mL) and FGF8b (100 ng/mL), and then cells were cultured for an additional 10 days. DA neuron-like cells were induced by withdrawing both Shh and FGF8b from NM. Over the next 25 days, reprogrammed cells began to change to neuron-like cell morphology. DA neuron-like cells were analyzed by immunofluorescence and semiquantitative RT-PCR.

Oh S.I., Park H.S., Hwang I., Park H.K., Choi K.A., Jeong H., Kim S.W, & Hong S. (2014). Efficient Reprogramming of Mouse Fibroblasts to Neuronal Cells including Dopaminergic Neurons. The Scientific World Journal, 2014, 957548.

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Directed Differentiation of iPSCs to Midbrain Neurons

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iPSCs used in this study were previously generated and characterized from two Parkinson’s disease patients harboring the p.A53T-α-synuclein mutation and a healthy subject (control, wild-type SNCA)^{22 (link)}. For directed differentiation, iPSCs were allowed to form embryoid bodies and neural induction was initiated by applying a dual SMAD inhibition protocol in the presence of Noggin and TGFβ inhibitor for generation of neural precursor cells (NPCs)^{22 (link)}. NPCs were expanded in DMEM/F12/B27/N2-medium supplemented with HEPES, Glutamax, non-essential amino acids [NEAA] and 20ug/ml FGF2. For neuronal differentiation, NPCs were dissociated with accutase and seeded onto poly-L-ornithine (20 μg/ml; Sigma-Aldrich)/laminin (5 μg/ml; Sigma-Aldrich)-coated dishes in DMEM/F12/ B27/N2-medium supplemented with 200 ng/ml human recombinant sonic hedgehog (SHH, R&D Systems) and 100 ng/ml murine recombinant fibroblast growth factor 8b (FGF-8b, R&D Systems) for 7 days in vitro (DIV). Cells were then replated in medium supplemented with 20 ng/ml brain-derived neurotrophic factor (BDNF, R&D Systems), 20 ng/ml glial cell-derived neurotrophic factor (GDNF, R&D Systems), 200 μM ascorbic acid (AA, Sigma-Aldrich) and 0.5 mM cyclic AMP (cAMP, Sigma- Aldrich). The medium was changed every 2 to 3 days for 2 weeks.

Antoniou N., Prodromidou K., Kouroupi G., Boumpoureka I., Samiotaki M., Panayotou G., Xilouri M., Kloukina I., Stefanis L., Grailhe R., Taoufik E, & Matsas R. (2022). High content screening and proteomic analysis identify a kinase inhibitor that rescues pathological phenotypes in a patient-derived model of Parkinson’s disease. NPJ Parkinson's Disease, 8, 15.

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Directed Differentiation of Neurotransmitter Neurons

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The glutamate and γ-aminobutyric acid (GABA)-secreting neurons were autonomously differentiated from the NPCs in neurobasal medium with N2 and B27 supplements (Invitrogen). The procedure for serotonergic neuron and dopaminergic neuron induction was modified from previous reports [11] (link), [15] (link), [16] (link). Briefly, the NPCs were adhered to 1% Matrigel-coated plates on D10. The formation of dopaminergic neurons was induced by the addition of 40 µg/mL FGF8b (R&D Systems) and 1 µM purmorphamine (Merck-Millipore, Billerica, MA, USA), a sonic hedgehog (Shh) signal activator [17] (link), from D11 to D15. The medium was refreshed every 2 days. To promote terminal neural differentiation, a cocktail containing 10 ng/mL brain-derived neurotrophic factor (BDNF, Peprotech, Rocky Hill, NJ, USA), 10 ng/mL glia cell line-derived neurotrophic factor (GDNF, Peprotech) and 5% B27 supplement (Invitrogen) in neurobasal media was provided from D16 [18] (link). Serotonergic neurons and dopaminergic neurons were examined by immunocytostaining on D25.

Shen S.C., Shen C.I., Lin H., Chen C.J., Chang C.Y., Chen S.M., Lee H.C., Lai P.S, & Su H.L. (2014). Susceptibility of Human Embryonic Stem Cell-Derived Neural Cells to Japanese Encephalitis Virus Infection. PLoS ONE, 9(12), e114990.

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Differentiation of Dopaminergic Neurons

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A protocol reported by Zhang and Zhang (2010) (link) was adopted for the differentiation of dopaminergic neurons. The neural tube-like rosettes were maintained in NIM medium supplemented with 50 ng/ml FGF8b (R&D, Minneapolis, USA) and 100 ng/ml SHH (R&D, Minneapolis, USA) from day 10 for one week. On the 17th day, the neuroepithelial progenitors were enriched and expanded in NIM containing FGF8b, SHH, B27 (Invitrogen) and ascorbic acid (Sigma) for another week. Then, the neural progenitor aggregates were dissociated to single cells and plated onto a culture surface pre-coated with laminin using conditioned NDM containing 200 μM ascorbic acid, 1.0 mΜ cAMP (Sigma), 1 ng/ml TGFβ3 (R&D), 10 ng/ml BDNF (R&D), 10 ng/ml GDNF (R&D) and Wnt3a -conditioned medium (1×) for 3 weeks. On the 44th day 44, FGF8b, SHH and Wnt3a conditioned medium was withdrawn and the cells were maintained in NDM with ascorbic acid, cAMP, TGFβ₃, BDNF and GDNF.

Zhang S.Z., Li H.F., Ma L.X., Qian W.J., Wang Z.F, & Wu Z.Y. (2015). Urine-derived induced pluripotent stem cells as a modeling tool for paroxysmal kinesigenic dyskinesia. Biology Open, 4(12), 1744-1752.

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Midbrain Neuron Differentiation from hiPSCs

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On day −1, 400,000 KOLF2-1J or SFC065 (kindly provided by the laboratory of C. Klein) hiPSC/cm² were seeded in Matrigel-coated 6-well plate wells in StemFlex medium supplemented with 10 μM RI. On day 0, medium was switched to Neurobasal containing 0.5x B27 supplement without vitamin A, 0.5x N2, GlutaMAX, Pen/strep, non-essential amino acids, and LDN193189 (500 nM, Sigma), SB431542 (10 μM, Tocris), SHH-C24II (200 ng/ml, Miltenyi Biotec), Purmorphamine (0.7 μM, Sigma) and 0.7 μM CHIR99021 (Stemcell Technologies). CHIR99021 concentration was raised to 3 μM from day 4 to day 11, moment at which it was withdrawn from the medium. LDN193189 (500 nM, Sigma), SB431542 (10 μM, Tocris), SHH-C24II (200 ng/ml, Miltenyi Biotec), Purmorphamine (0.7 μM, Sigma) were withdrawn from the medium at day 7 and FGF8b (100 ng/mL, R&D Systems) was introduced from day 9 until day 16. At day 11, medium was shifted to Neurobasal-A medium (Life Technologies; 10888-022) supplemented with 1x B27 without Vit. A (Life Technologies; 12587010), 1x GlutaMAX (Life Technologies; 35050-038), 1x PenStrep (Life Technologies; 15140-122), 10 ng/mL BDNF (R&D Systems; 248-BDB-050/CF), 10 ng/mL GDNF (R&D Systems; 212-GD-010), 200 μM ascorbic acid, 0.5 mM dbcAMP (Sigma-Aldrich), 10 μM DAPT (Tocris; 2634). On day 17, ventral midbrain neural progenitors were cryopreserved.

Pantazis C.B., Yang A., Lara E., McDonough J.A., Blauwendraat C., Peng L., Oguro H., Kanaujiya J., Zou J., Sebesta D., Pratt G., Cross E., Blockwick J., Buxton P., Kinner-Bibeau L., Medura C., Tompkins C., Hughes S., Santiana M., Faghri F., Nalls M.A., Vitale D., Ballard S., Qi Y.A., Ramos D.M., Anderson K.M., Stadler J., Narayan P., Papademetriou J., Reilly L., Nelson M.P., Aggarwal S., Rosen L.U., Kirwan P., Pisupati V., Coon S.L., Scholz S.W., Priebe T., Öttl M., Dong J., Meijer M., Janssen L.J., Lourenco V.S., van der Kant R., Crusius D., Paquet D., Raulin A.C., Bu G., Held A., Wainger B.J., Gabriele R.M., Casey J.M., Wray S., Abu-Bonsrah D., Parish C.L., Beccari M.S., Cleveland D.W., Li E., Rose I.V., Kampmann M., Aristoy C.C., Verstreken P., Heinrich L., Chen M.Y., Schüle B., Dou D., Holzbaur E.L., Zanellati M.C., Basundra R., Deshmukh M., Cohen S., Khanna R., Raman M., Nevin Z.S., Matia M., Van Lent J., Timmerman V., Conklin B.R., Chase K.J., Zhang K., Funes S., Bosco D.A., Erlebach L., Welzer M., Kronenberg-Versteeg D., Lyu G., Arenas E., Coccia E., Sarrafha L., Ahfeldt T., Marioni J.C., Skarnes W.C., Cookson M.R., Ward M.E, & Merkle F.T. (2022). A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell stem cell, 29(12), 1685-1702.e22.

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