Recombinant human gdnf

Manufactured by R&D Systems

Sourced in United States

Recombinant human GDNF is a growth factor protein produced in a laboratory setting. It is a soluble, glycosylated protein that is composed of two identical subunits.

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Lab products found in correlation

Tetracycline, by Merck Group (4 mentions) Recombinant rat gfrα1, by R&D Systems (2 mentions) Trypsin edta, by Thermo Fisher Scientific (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Fibronectin, by Merck Group (2 mentions) Resazurin sodium salt, by Merck Group (2 mentions) Hoechst bisbenzimide h 33342, by Merck Group (2 mentions) Tween 20, by Carl Roth (2 mentions) Recombinant human fgf 2, by R&D Systems (2 mentions) Cd90.2 beads, by Miltenyi Biotec (1 mentions) Recombinant human fgf9, by R&D Systems (1 mentions) Gw 788388, by MedChemExpress (1 mentions) Tofacitinib citrate, by MedChemExpress (1 mentions) Sb203580, by R&D Systems (1 mentions) Putrescine, by Merck Group (1 mentions) Holo transferrin, by Merck Group (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) α mem, by Thermo Fisher Scientific (1 mentions) Recombinant human bfgf, by R&D Systems (1 mentions)

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Human GDNF (5 citations)

10 protocols using recombinant human gdnf

Culturing THY-1+ Germ Cells from Murine Testes

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THY‐1⁺ germ cell cultures were established as described previously.⁸ Briefly, male C57 LacZ pups (5‐8 days post‐partum) were sacrificed and testes were digested using Trypsin‐EDTA (Gibco, USA). Cells were magnetically sorted using CD90.2 beads (Miltenyi, USA) and plated onto Sandos inbred mouse (SIM)–derived 6‐thioguanine‐resistant and ouabain‐resistant (STO) feeders (SNL76/7, ATCC). Germ cell cultures were maintained in a humidified atmosphere at 37°C contains 5% CO₂ with Mouse serum‐free media (mSFM) with 20 ng/mL recombinant human GDNF (R&D, USA), 150 ng/mL recombinant rat GFRα1 (R&D) and 1 ng/mL recombinant human FGF2 (Corning, USA). Other reagents were used as indicated: recombinant human FGF9 (R&D), SB203580, GW788388 and Tofacitinib citrate (all from MedChemExpress, USA). Germ cell counts were determined with a hemocytometer. Feeder cells were identified and discounted from counts based on morphology.²² SSC number was calculated using the following formula, adapted from reference ⁴:

S S C g r o w t h = \frac{Cellsharvested}{Cellsseeded} \times \frac{Colonies}{10^{5} c e l l s t r a n s p l a n t e d}

Here, ‘cells seeded’ indicates the number of cultured THY‐1⁺ germ cells seeded at the beginning of each time period and ‘cells harvested’ is the cell count at the end. ‘Colonies’ indicates the number of distinct colonies counted per testis and is divided by the number of cultured THY‐1⁺ germ cells transplanted per testis.

Yang F., Whelan E.C., Guan X., Deng B., Wang S., Sun J., Avarbock M.R., Wu X, & Brinster R.L. (2020). FGF9 promotes mouse spermatogonial stem cell proliferation mediated by p38 MAPK signalling. Cell Proliferation, 54(1), e12933.

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Directed Differentiation of hESCs

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Colonies of HS401 cells were collected by incubation with 1 mg/ml Collagenase IV (Worthington Biochemical Corporation) for 40 min in 37°C and plated as small clumps on SNL feeders in 1:3 ratio using mTeSR1 medium. The next day, medium was changed to SSC-differentiation medium [5 (link)] consisting of basal medium α-MEM (Life Technologies), 0.2% w/v bovine serum albumin (BSA, Sigma), 1x Glutamax (Life Technologies), 10 mM HEPES (Sigma), 50 U/ml and 50 mg/ml penicillin-streptomycin, 50 μM β-mercaptoethanol (Life Technologies), 5 μg/ml human recombinant insulin (Sigma), 10 μg/ml holo-transferrin (Sigma), 30 nM sodium selenite (Sigma), 60 μM putrescine (Sigma), 2.36 μM palmitic acid (Sigma), 0.21 μM palmit oleic acid (Sigma), 0.88 μM stearic acid (Sigma), 1.02 μM oleic acid (Sigma), 2.71 μM linoleic acid (Sigma), 0.43 μM linolenic acid (Sigma), 1 ng/ml human recombinant bFGF (R&D Systems) and 20 ng/ml recombinant human GDNF (R&D Systems). The SSC-differentiation medium was gassed with 90% N₂, 5% CO_2, 5% O₂ gas mixture for 30s before changed to cells every two days.

Panula S., Reda A., Stukenborg J.B., Ramathal C., Sukhwani M., Albalushi H., Edsgärd D., Nakamura M., Söder O., Orwig K.E., Yamanaka S., Reijo Pera R.A, & Hovatta O. (2016). Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells. PLoS ONE, 11(10), e0165268.

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LUHMES Cell Differentiation Protocol

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LUHMES cells were a kind gift from Prof. Marcel Leist and were cultured and differentiated as described previously (9 (link),80 (link),95 (link)); the methods below are reproduced from (9 (link)) with permission. Briefly, proliferating cells were cultured in Advanced Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with N2 (Gibco), 2 mM l-glutamine (Gibco), and 40 ng/mL recombinant basic fibroblast growth factor (bFGF, R&D Systems Inc., Minneapolis, MN, USA), in pre-coated flasks (Corning Inc., Corning, NY, USA) with 50 μg/mL PLO (Sigma-Aldrich, St. Louis, MO, USA), at 37°C in a humidified 5% CO₂ atmosphere. Differentiation was achieved by replacing the proliferating medium with DMEM/F12 supplemented with N2 (Gibco), 2 mM l-glutamine (Gibco), 1 μg/mL tetracycline (Sigma-Aldrich), 1 mM dibutyryl cAMP (cAMP; Sigma-Aldrich), and 2 ng/mL recombinant human GDNF (R&D Systems). After 2 days in culture (pre-differentiation), cells were trypsinized and seeded into plates pre-coated with 50 μg/mL PLO and 1 μg/mL fibronectin (Sigma-Aldrich). On day 5 of differentiation, one third of the culture medium was replaced with fresh medium. Cells were harvested after 8 days of differentiation.

Schaffner S.L., Wassouf Z., Lazaro D.F., Xylaki M., Gladish N., Lin D.T., MacIsaac J., Ramadori K., Hentrich T., Schulze-Hentrich J.M., Outeiro T.F, & Kobor M.S. (2022). Alpha-synuclein overexpression induces epigenomic dysregulation of glutamate signaling and locomotor pathways. Human Molecular Genetics, 31(21), 3694-3714.

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Derivation of Adult Stem Cell Lines

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To derive adult SSCs lines from adult C57BL/6 and Col1a1-4F2A-OSKM mice, α6⁺/c-Kit^-/low/β2m^- germinal cells were isolated and maintained on mitomycin-C-treated MEFs as previously described [18 ]. The SSCs culture medium was composed of Stem Span (Stemcell Technologies) and B27 supplement (Life Technologies) and supplemented with recombinant human GDNF (40 ng·ml^-1, R&D Systems), recombinant rat GFRα1 (300 ng·ml^-1, R&D Systems), FGF2 (1 ng·ml^-1, Life Technologies), and ES-Cult™ Foetal Bovine Serum (1%, Stemcell Technologies). Every 3-4 days, SSCs clusters were split using enzymatic digestion with 0.05% trypsin-EDTA (Life Technologies). SSCs were used for reprogramming after at least 1.5 months of culture. MEF cultures were established by trypsin digestion of Col1a1-4F2A-OSKM 13.5 dpc embryos, and the resulting cells were cultured in DMEM supplemented with 10% FBS, L-glutamine and penicillin/streptomycin.

Corbineau S., Lassalle B., Givelet M., Souissi-Sarahoui I., Firlej V., Romeo P.H., Allemand I., Riou L, & Fouchet P. (2016). Spermatogonial stem cells and progenitors are refractory to reprogramming to pluripotency by the transcription factors Oct3/4, c-Myc, Sox2 and Klf4. Oncotarget, 8(6), 10050-10063.

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Biofunctionalized Organosilica Co-culture

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tRFP-overexpressing LUHMES were differentiated in LUHMES differentiation medium Advanced DMEM/F12, 1× N2 supplement, 2 mM l-glutamine, 1 mM dbcAMP (Sigma), 1 μg mL⁻¹ tetracycline (Sigma) and 2 ng mL⁻¹ recombinant human GDNF (R+D Systems). After 2 days, cells were enzymatically dissociated with trypsin, collected in Advanced DMEM/F12 and centrifuged at 300×g for 5 min at RT. The cell pellet was resuspended in differentiation medium and 2 × 10⁶ cells were seeded onto one side of biofunctionalized bidirectional structural gradient organosilica material.
18 h after seeding of LUHMES cells, HeLa cells that were pre-stained for 30 min with Calcein-AM (1 μM) were seeded on the other side of the organosilica material. After another 6 h, imaging was performed at excitation wavelengths 350 nm, 488 nm and 535 nm.

Bronner H., Holzer A.K., Finke A., Kunkel M., Marx A., Leist M, & Polarz S. (2020). The influence of structural gradients in large pore organosilica materials on the capabilities for hosting cellular communities. RSC Advances, 10(29), 17327-17335.

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Chemically Defined Cell Culture Components

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Dibutyryl-cAMP (cAMP), fibronectin, Hoechst bisbenzimide H-33342, resazurin sodium salt, tetracycline, L-cysteine and reduced glutathione (GSH) were purchased from Sigma (Steinheim, Germany). Recombinant human FGF-2 and recombinant human GDNF were purchased from R&D Systems (Minneapolis, USA). Tween-20 and sodium dodecyl sulphate (SDS) were purchased from Roth (Karlsruhe, Germany). All cell culture reagents were purchased from Gibco/Fisher Scientific (Hampton, New Hampshire, USA) unless otherwise specified. MG-132 was purchased from Selleckchem (Houston, USA).

Gutbier S., Spreng A.S., Delp J., Schildknecht S., Karreman C., Suciu I., Brunner T., Groettrup M, & Leist M. (2018). Prevention of neuronal apoptosis by astrocytes through thiol-mediated stress response modulation and accelerated recovery from proteotoxic stress. Cell Death and Differentiation, 25(12), 2101-2117.

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Neuronal Cell Culture Reagents

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Dibutyryl-cAMP (cAMP), fibronectin, hoechst bisbenzimide H-33342, resazurin sodium salt, tetracycline, tetramethylrhodamine ethyl ester (TMRE) and MPP⁺ were from Sigma (Steinheim, Germany). Recombinant human FGF-2 and recombinant human GDNF were from R&D Systems (Minneapolis, MN, USA). Tween-20 and sodium dodecyl sulfate (SDS) were from Roth (Karlsruhe, Germany). All culture reagents were from Gibco (Carlsbad, CA, USA) unless otherwise specified.

Krug A.K., Gutbier S., Zhao L., Pöltl D., Kullmann C., Ivanova V., Förster S., Jagtap S., Meiser J., Leparc G., Schildknecht S., Adam M., Hiller K., Farhan H., Brunner T., Hartung T., Sachinidis A, & Leist M. (2014). Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP+. Cell Death & Disease, 5(5), e1222-.

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Differentiation of LUHMES Cells into Neurons

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Lund Human Mesencephalic (LUHMES) cells were obtained, maintained and differentiated as previously described[23 (link)]. Briefly, proliferating LUHMES cells were cultured in Advanced Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Gibco) supplemented with 1xN2 (Gibco), 2 mM L-glutamine (Gibco) and 40 ng/mL recombinant basic fibroblast growth factor (R&D Systems). For differentiation, proliferation medium was replaced by DMEM/F12 containing 1xN2, 2 mM L-glutamine, 1 mM dibutyryl cAMP (Sigma Aldrich), 1 μg/mL tetracyclin (Sigma-Aldrich) and 2 ng/mL recombinant human GDNF (R&D Systems). Two days (D2) after adding differentiation medium, cells were seeded in plates (Nunclon) pre-coated with 50 μg/mL poly-L-ornithine and 1 μg/mL fibronectin (Sigma-Aldrich), and grown at 37°C in humidified 5% CO₂ atmosphere.

Pinho R., Guedes L.C., Soreq L., Lobo P.P., Mestre T., Coelho M., Rosa M.M., Gonçalves N., Wales P., Mendes T., Gerhardt E., Fahlbusch C., Bonifati V., Bonin M., Miltenberger-Miltényi G., Borovecki F., Soreq H., Ferreira J.J, & F. Outeiro T. (2016). Gene Expression Differences in Peripheral Blood of Parkinson’s Disease Patients with Distinct Progression Profiles. PLoS ONE, 11(6), e0157852.

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Synthesis and Preparation of RET Inhibitor SPP86

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The RET inhibitor SPP86 was synthesized by a literature procedure [45 (link)]. Stock solutions of SPP86 (10 mM) in DMSO were stored at 4°C and diluted just prior to use. 17-β estradiol (E2), 4- hydroxy tamoxifen (4-OHT) and insulin were obtained from Sigma-Aldrich (Stockholm, Sweden) dissolved in ethanol and stored at 4°C. PF-573228 was from Tocris Bioscience (Bristol, United Kingdom), dissolved in DMSO and stored at -20°C. ICI182,780 was from Tocris Bioscience dissolved in ethanol and stored at -20°C. Sorafenib (BAY43-9006) was obtained from AH Diagnostics AB (Skärholmen, Sweden) and stock solutions in DMSO were stored at -20°C. Recombinant human GDNF was obtained from R&D systems (Abingdon, United Kingdom) and was reconstituted and stored according to the supplier’s instructions.

Alao J.P., Michlikova S., Dinér P., Grøtli M, & Sunnerhagen P. (2014). Selective inhibition of RET mediated cell proliferation in vitro by the kinase inhibitor SPP86. BMC Cancer, 14, 853.

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Evaluating GDNF Effects on Human Oocyte Maturation

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Based on a previous study (13) and the results of our preliminary experiments (Supplemental Table 2), we chose the concentration of 50 nM to evaluate the effects of GDNF on human oocyte maturation in the in vitro study. A total of 200 GV-stage COCs were randomly transferred into the G-IVF (Vitrolife, Sweden AB, Sweden) medium supplemented with 10% synthesis serum substituent with (n = 100) or without (n = 100) 50 nM of recombinant human GDNF (R&D Systems, Minneapolis, MN) and were incubated at 37°C in a controlled atmosphere of 5% O 2 , 6% CO 2 , and 89% N 2 for 24 hours. For measurement of oocyte maturity, COCs were denuded of CCs by repeated pipetting. Oocytes without the first polar body are regarded as immature and were classified as GV stage or metaphase I (MI) stage according to the presence of a GV. Oocytes that exhibit extrusion of the first polar body (MII stage) were fertilized by intracytoplasmic sperm injection (ICSI) using sperm with normal semen parameters. Semen analysis was performed according to the 2010 World Health Organization laboratory manual (23) . Fertilization was assessed by the appearance of two pronuclei (2PN) in the zygote 16 to 18 hours after microinjection. Embryos were cultured and observed every day until reaching the blastocyst stage (approximately 5 to 6 days).

Cui L., Fang L., Mao X., Chang H.M., Leung P.C.K, & Ye Y. (2018). GDNF-Induced Downregulation of miR-145-5p Enhances Human Oocyte Maturation and Cumulus Cell Viability. The Journal of clinical endocrinology and metabolism, 103(7).

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