The procedures for isolating and culturing DRG neurons have been described previously [17 (link),18 (link)], and these procedures were followed with some modifications. Briefly, timed–pregnant NIH Swiss mice (G15) were euthanized with CO2. The DRGs were dissected from the embryos and placed in a tube containing Hibernate–E solution (BrainBits LLC, USA) at 4°C. The DRGs were then incubated in Krebs buffer containing 0.05% trypsin for 30 min at 37°C. The digestion was terminated by the addition of 0.2% trypsin inhibitor (Sigma, USA) and 0.02% DNase.The solution was then triturated gently until a cloudy suspension appeared. The cells were washed once with Krebs buffer and then resuspended in growth medium that consisted of Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, USA) supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, 100 ng⁄ml 7S nerve growth factor (Sigma, USA), 80 μM 5-fluoro-2′–deoxyuridine, and 100 μM uridine. Approximately 0.2–0.3×105 cells⁄ml were placed on polylysine–coated coverslips. The cells were grown at 37°C in an atmospheric environment containing 95% O2 and 5% CO2, and the media was refreshed every 3 days.
7s nerve growth factor
Manufactured by Merck Group
Sourced in United States
7S nerve growth factor is a laboratory product developed by Merck Group. It is a protein complex that plays a central role in the survival and growth of certain types of nerve cells. The 7S nerve growth factor provides a fundamental biological function, but its specific intended uses are not included in this factual description.
Automatically generated - may contain errors
Lab products found in correlation
Draq5, by Cell Signaling Technology (2 mentions)
Trypsin inhibitor, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
Poly d lysine pdl, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Snd450, by Microfluidics (1 mentions)
Glutamine, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Thiazolyl blue, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dmem low glucose medium, by Merck Group (1 mentions)
Aβ42 peptide, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Horse serum, by HiMedia (1 mentions)
Chondrocyte differentiation medium, by Merck Group (1 mentions)
7 protocols using 7s nerve growth factor
1
Isolation and Culture of DRG Neurons
2
Induction of Neuronal PC12 Cells
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PC12 was purchased from Shanghai cell bank of Chinese Academy of Sciences. PC12 was revived and then cultivated with 10% FBS 1×P.S. 1640 culture medium (C11875500BT, GIBCO, USA) at 37°C as well as incubator with 5% CO2. 10 nM 7S nerve growth factor (Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium to induce PC12 cells to neuronal PC12 cells. The cells were co-cultured with the nerve growth factor for 3 days. When the cell density reached about 80%, the cells were cleaned with PBS (pH 7.4), and digested with 1ml 0.25% pancreatin, which was suctioned and abandoned after affecting for 2-3min, then 3ml preheating DEME complete medium at 37°C was added. The bottle bottom was gently blown until the cells were blown down. High-speed centrifuge was used to remove cell suspension. Cell precipitation was collected and added 6ml DEME complete medium for the resuspension of cells. The cell suspension was inoculated into 3 new culture bottles (2ml/bottle) and replenished with 3ml DEME complete medium, which was cultured with 5% CO2 at 37°C and saturated humidity. The subcultured cells were divided into control group (normal cultured PC12) and case group (OGSD 12h/R 0-6h).
3
Rat DRG Neuron Culture Preparation
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Neuronal cultures were prepared as previously described [4 (link),21 (link),22 (link)]. Briefly, standard neuron devices (SND450; Xona Microfluidics, USA), plasma bonded to cover glasses, were coated with 0.5 mg/ml poly-D-lysine (PDL; Sigma) in borate buffer (pH 8.5), and laminin (10 μg/ml; Sigma) at 37°C with 5% CO2. Rat DRG dissociated neurons were prepared as described above, and a 4 μl suspension containing approximately 40,000 neurons was added to the cell body compartment of the devices (S1 Fig ). Neurons were grown in neurobasal medium supplemented with L-glutamine (4 mM; Invitrogen), B-27 supplement (2%; Life Technologies), brain-derived neurotrophic factor (BDNF; 5 ng/ml; Sigma) and 7S nerve growth factor (100 ng/ml; Sigma) for 3 days at 37°C with 5% CO2 to allow the axons to grow into the axonal compartment of the device prior to infection (S1 Fig ).
4
Dorsal Root Ganglia Neuron Isolation
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Dorsal root ganglia (DRG) were obtained by microdissection from White Leghorn chicken embryos (Sinyavino Poultry, Leningrad District, Russia). Experiments were performed on cultured isolated DRG neurons of 10-12-day chicken embryos.
Culture of Dissociated DRG DRG were dissected from the L5-S1 regions of 10-12-day-old chicken embryos and dissociated by mechanical pipetting under physiological conditions. The culture fluid was added to the cell suspension in order to obtain the desired cell density in a plastic Petri dish. The non-neuronal cells were removed by allowing them to settle onto the surfaces of plastic 90-mm Petri dishes for 25 min at 37 °C. The medium did not contain any proteolytic enzymes and consisted of 45% Hank's solution, 40% Eagle's medium, and 15% fetal calf serum, and was supplemented with insulin (0.5 U/ml), glucose (0.6%), glutamine (2 mM), gentamicin (100 U/ml), and 7S nerve growth factor (10 ng/ml) (Sigma). The cell suspension was plated onto fibronectin-and collagen-coated 40-mm Petri dishes. Their bottoms were covered with 7 µg/ml solution of collagen. After an hour, the formed collagen film was coated with fibronectin (10 µg/ml). The cells were cultured for three days at 37 °C and 5% CO 2 . Ouabain was added to the cell culture medium. The cells cultured without ouabain were used as the control.
Culture of Dissociated DRG DRG were dissected from the L5-S1 regions of 10-12-day-old chicken embryos and dissociated by mechanical pipetting under physiological conditions. The culture fluid was added to the cell suspension in order to obtain the desired cell density in a plastic Petri dish. The non-neuronal cells were removed by allowing them to settle onto the surfaces of plastic 90-mm Petri dishes for 25 min at 37 °C. The medium did not contain any proteolytic enzymes and consisted of 45% Hank's solution, 40% Eagle's medium, and 15% fetal calf serum, and was supplemented with insulin (0.5 U/ml), glucose (0.6%), glutamine (2 mM), gentamicin (100 U/ml), and 7S nerve growth factor (10 ng/ml) (Sigma). The cell suspension was plated onto fibronectin-and collagen-coated 40-mm Petri dishes. Their bottoms were covered with 7 µg/ml solution of collagen. After an hour, the formed collagen film was coated with fibronectin (10 µg/ml). The cells were cultured for three days at 37 °C and 5% CO 2 . Ouabain was added to the cell culture medium. The cells cultured without ouabain were used as the control.
5
Fabrication and Characterization of Gold Nanoparticles
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Gold nanospheres 5 nm (product code # 752568), 50 nm (# 753645) and 100 nm (# 753688) as well as Aβ42 peptide were purchased from Sigma Aldrich, USA. The AuNSs were stored at 4 °C and used as supplied. Ni–NTA agarose was obtained from Qiagen. IPTG and PMSF were purchased from Sigma Aldrich, USA. Urea was purchased from Calbiochem, India. All other chemicals were of analytical grade and were procured from Merck, India. Horse Serum was bought from Himedia Laboratories. Fetal Bovine Serum (FBS), trypsin–EDTA, penicillin and sterptomycin were obtained from Gibco (Thermo Fisher Scientific). Thiazolyl Blue, tetrazolium bromide (MTT), DMEM low glucose medium and Nerve Growth Factor-7S were purchased from Sigma Aldrich.
6
Differentiation of EC ID3+ Stem Cells
7
Chondrocyte Differentiation of NRF1 MCF10A
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For differentiation, both an NRF1 MCF10A clone and the vector MCF10A were maintained in a chondrocyte differentiation medium (Sigma, St. Louis, MO, USA) for 28 days. The NRF1 MCF10A clone and vector MCF10A were cultured in either SMGS plus DMEM-F12 or neurobasal media plus nerve growth factor-7S (Sigma), Glutamax, and N2 supplement for a total of 28 days. We determined cell differentiation of NRF1 MCF10A clone at 28 days with staining of Alician blue, α-smooth muscle actin (smooth muscle marker), and β-tubulin III (neuron marker). Nuclei were counterstained with DRAQ5® (Cell Signaling).
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