Recombinant human gdnf

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Recombinant human GDNF is a purified protein that functions as a neurotrophic factor. It is produced in a mammalian cell expression system.

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17 protocols using recombinant human gdnf

Optimized Culture Media for Stem Cell Growth

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The culture medium consisted of StemPro‐34 SFM with Stem Pro supplement (Kanatsu‐Shinohara, Ogonuki, et al., 2003; Wu et al., 2015; Zhang et al., 2014; Gibco, #10639–011), 25 μg/mL insulin (Sigma, #I1882), 100 μg/mL transferrin (Sigma, #T1428), 60 μM putrescine (Sigma, #P5780), 30 nM sodium selenite (Sigma, #S5261), 6 mg/mL D‐(+)‐glucose (Sigma, #G7021), 30 μg/mL pyruvic acid (Sigma, #P4562), 1 μL/mL DL‐lactic acid (Sigma, #L7900), 5 mg/mL bovine serum albumin (Calbiochem, #126609), 2 mM L‐glutamine (Millipore, #TMS‐002‐C), 100x β‐mercaptoethanol (Specialty Media, #ES‐007‐E), minimal essential medium vitamin solution (Gibco, #11120–052), 100x nonessential amino acid solution (Gibco, #11140–035), 1% penicillin/streptomycin (Specialty Media, #TMS‐AB2‐C), 0.1 mM ascorbic acid (Sigma, #A4034), 10 μg/mL d‐biotin (Sigma, #B4639), 20 ng/mL recombinant human epidermal growth factor (Gibco, #PMG8041), 10 ng/mL recombinant human basic fibroblast growth factor (PeproTech, #AF‐100‐18B‐250), 10 ng/mL recombinant human GDNF (PeproTech, #450–10‐250), 1 IU/mL Leukemia Inhibitory Factor (Millipore, #ESG1107), and 1% fetal bovine serum (HyClone, #SH30071.03). Cells were maintained at 37°C with 5% CO₂.

Zhou H., Zeng Z., Koentgen F., Khan M, & Mombaerts P. (2019). The testicular soma of Tsc22d3 knockout mice supports spermatogenesis and germline transmission from spermatogonial stem cell lines upon transplantation. Genesis (New York, N.y. : 2000), 57(6), e23295.

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Protein Labeling with Dylight-488

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Dylight-488 NHS-ester (Pierce) was dissolved in dimethyl formamide at 10 mg/mL. Recombinant human GDNF (Peprotech, Rocky Hill, NJ) was dissolved in 8 mM sodium phosphate buffer (pH 7.4). Dylight-488 was added to the solution for a final GDNF concentration of 10 μg/mL and a final Dylight-488 concentration of 50 ng/mL and incubated overnight at 2°C. The solution was then dialyzed using Slide-A-Lyzer MINI Dialysis Units (Thermo Scientific, Rockford, IL, 3500 MWCO) in 8 mM sodium phosphate buffer (pH 7.4) to remove unbound Dylight-488.

Roam J.L., Nguyen P.K, & Elbert D.L. (2014). Controlled Release and Gradient Formation of Human Glial-Cell Derived Neurotrophic Factor from Heparinated Poly(ethylene glycol) Microsphere-based Scaffolds. Biomaterials, 35(24), 6473-6481.

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Fluorescent Labeling of GDNF Protein

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Example 15

Dylight-488 NHS-ester (Pierce) was dissolved in dimethyl formamide at 10 mg/mL. Recombinant human GDNF (Peprotech, Rocky Hill, N.J.) was dissolved in 8 mM sodium phosphate buffer (pH 7.4). Dylight-488 was added to the solution for a final GDNF concentration of 10 mg/mL and a final Dylight-488 concentration of 50 ng/mL and incubated overnight at 4° C. The solution was then dialyzed using Slide-A-Lyzer MINI Dialysis Units (Thermo Scientific, Rockford, Ill., 3500 MWCO) in 8 mM sodium phosphate buffer (pH 7.4) to remove unbound Dylight-488.

US10137082B2. Hydrogel microparticle scaffold with gradients of degradability and methods thereof (2018-11-27). None [US], None [US]. Inventors: Donald L. Elbert [US], Jacob Roam [US].

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Establishing Mouse Germline Stem Cell Culture

Cited 2 times

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Mouse GSCs were cultured as previously reported (25 (link), 27 (link), 29 (link)). The cells were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs; Gibco, A34962), using a medium (medium I) composed of StemPro-34 SFM medium (Thermo Fisher Scientific), StemPro-34 Supplement (Thermo Fisher Scientific), 1% fetal bovine serum (FBS), recombinant human GDNF (10 ng/ml, 450-10, Peprotech), recombinant human bFGF (10 ng/ml, 100-18B, Peprotech), recombinant human EGF (20 ng/ml, AF-100-15, Peprotech), recombinant human LIF (10 ng/ml, CYT-644, Prospec), as well as other components as previously reported (29 (link)) and described in Supplementary Table 1. The cells were refreshed every 2-3 days, and passaged every 5-7 days at a ratio of 1:4-6 on freshly plated mitotically inactivated mouse embryonic fibroblasts. The cells were maintained at 37°C in 5% CO2 in air.
As a feeder cell to support in vitro meiosis of mouse mGSCs, we used an available immortalized Sertoli cell lines SK49 (30 (link)). The cells were cultured at 37°C and 5% CO2 in Dulbecco’s Eagle’s medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL).

Lei Q., Zhang E., van Pelt A.M, & Hamer G. (2021). Meiotic Chromosome Synapsis and XY-Body Formation In Vitro. Frontiers in Endocrinology, 12, 761249.

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Porcine Spermatogonial Stem Cell Culture

Cited 1 time

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The cell line used in this study was the immortalized porcine SSC line established by us [31 (link)]. The cell line was characterized by immunofluorescence staining of different SSC markers. The cells were cultured in DMEM/high glucose (Gibco, Grand Island, NY, USA) containing 5% fetal bovine serum (Gibco, Mesenchymal Stem Cell FBS Qualified), 5% knockout serum replacement (KSR; Gibco), 100 unit/mL penicillin and streptomycin (Hyclone, Logan, Utah), 1 × MEM vitamin solution (Gibco), 1× non-essential amino acid (NEAA; Gibco), 2 mmol/L Glutamax (Gibco), 40 ng/mL recombinant human GFRA1 (BioLegend, San Diego, CA, USA), 20 ng/mL recombinant human GDNF (Peprotech, Rocky Hill, NJ, USA) and 10 ng/mL recombinant human bFGF (Peprotech). The cells were maintained at 37 ℃ in a humidified incubator containing 5% CO₂.

Liu R., Liu Z., Guo M., Zeng W, & Zheng Y. (2022). SETDB1 Regulates Porcine Spermatogonial Adhesion and Proliferation through Modulating MMP3/10 Transcription. Cells, 11(3), 370.

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GDNF Microparticle Loading Protocol

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Example 14

Recombinant human GDNF (Peprotech, Rocky Hill, N.J.) was dissolved in 8 mM sodium phosphate buffer (pH 7.4) and added to washed microparticles such that the GDNF concentration within the supernatant was 250 ng/mL (note higher concentrations used for DRG experiments below). The microparticle/GDNF solution was well mixed by tituration and incubated 2 h at 4° C. to allow diffusion of GDNF into the microparticles. Immediately before scaffold formation, the microparticles were centrifuged at 14,100 g, supernatant was removed, and microparticles were re-suspended in 8 mM sodium phosphate.

US10682309B2. Hydrogel microparticle scaffold with gradients of degradability and methods thereof (2020-06-16). None [US], None [US]. Inventors: Donald L. Elbert [US], Jacob Roam [US].

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Fluorescent Labeling of GDNF Protein

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Example 15

US10682309B2. Hydrogel microparticle scaffold with gradients of degradability and methods thereof (2020-06-16). None [US], None [US]. Inventors: Donald L. Elbert [US], Jacob Roam [US].

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Fluorescent GDNF Conjugation Protocol

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Dylight-488 NHS-ester (Pierce) was dissolved in dimethyl formamide at 10 mg/mL. Recombinant human GDNF (Peprotech, Rocky Hill, NJ) was dissolved in 8 mM sodium phosphate buffer (pH 7.4). Dylight-488 was added to the solution for a final GDNF concentration of 10 μg/mL and a final Dylight-488 concentration of 50 ng/mL and incubated overnight at 4°C. The solution was then dialyzed using Slide-A-Lyzer MINI Dialysis Units (Thermo Scientific, Rockford, IL, 3500 MWCO) in 8 mM sodium phosphate buffer (pH 7.4) to remove unbound Dylight-488.

Roam J.L., Yan Y., Nguyen P.K., Kinstlinger I.S., Leuchter M.K., Hunter D.A., Wood M.D, & Elbert D.L. (2015). A Modular, Plasmin-Sensitive, Clickable Poly(ethylene glycol)-Heparin-Laminin Microsphere System for Establishing Growth Factor Gradients in Nerve Guidance Conduits. Biomaterials, 72, 112-124.

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Encapsulation of GDNF in Microspheres

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Recombinant human GDNF (Peprotech, Rocky Hill, NJ) was dissolved in 8 mM sodium phosphate buffer (pH 7.4) and added to washed microspheres such that the GDNF concentration within the supernatant was 250 ng/mL (note higher concentrations used for DRG experiments below). The microsphere/GDNF solution was well mixed by tituration and incubated 2 h at 4°C to allow diffusion of GDNF into the microspheres. Immediately before scaffold formation, the microspheres were centrifuged at 14,100 g, supernatant was removed, and microspheres were re-suspended in 8 mM sodium phosphate.

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Porcine Spermatogonial Stem Cell Culture

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The sorted immortalized porcine SSCs were cultured in the complete medium made up of DMEM (high glucose; Thermo Fisher Scientific), 5% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific), 5% (v/v) knockout serum replacement (KSR; Thermo Fisher Scientific), 2 mmol/L Glutamax (Thermo Fisher Scientific), 1× non-essential amino acid (Thermo Fisher Scientific), 1 × vitamin solution (Thermo Fisher Scientific), 5 × 10^− 5 mol/L 2-mercaptoethanol (Sigma-Aldrich), 1× penicillin-streptomycin (Hyclone), 20 ng/mL recombinant human GDNF (Peprotech), 40 ng/mL soluble GFRA1 (Peprotech) and 10 ng/mL recombinant human bFGF (Peprotech). The cells were refreshed every 2 d, and when reaching 80% of confluency, they were passaged to new wells coated with laminin (20 μg/mL; Sigma-Aldrich) at a ratio of 1: 2. The cells were maintained at 35 °C in an atmosphere of 5% CO₂ in air. For retinoic acid (RA)-induced differentiation, cells were exposed to the growth factor-free medium harboring 5 μmol/L all-trans-RA (Sigma-Aldrich) for 2 d. In control groups, 0.1% ethanol was added to the medium as a vehicle.

Zheng Y., Feng T., Zhang P., Lei P., Li F, & Zeng W. (2020). Establishment of cell lines with porcine spermatogonial stem cell properties. Journal of Animal Science and Biotechnology, 11, 33.

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