PC12 cells (pheochromocytoma cells derived from the adrenal gland of Rattus norvegicus) (ATCC, Manassas, VA), were grown in 75-cm2 culture flasks at 37°C in Dulbecco’s Modified Eagle’s Medium (DMEM) (4.5 g/L glucose, L-glutamine, without pyruvate), supplemented with 10% bovine calf serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) in 10% CO2. For NGF treatment, PC12 cells were treated with 100 ng/mL of NGF (Sigma-Aldrich, St. Louis, MO) dissolved in complete media for three consecutive days. Control cells without NGF were also grown under the same conditions. For quantitative assessment of neurite outgrowth, PC12 cells were only treated with NGF for 2 days instead of 3, given that the density of neurite outgrowth does not allow for proper tracing of neurites belonging to a specific cell body.
Nerve growth factor (ngf)
Manufactured by Merck Group
Sourced in United States, Germany, Italy, Austria, China, United Kingdom, Japan, Singapore
NGF is a laboratory instrument used for the detection and quantification of nerve growth factor (NGF) in biological samples. It functions as a tool for researchers studying neurological processes and disorders related to NGF. The core purpose of NGF is to provide accurate and reliable measurements of NGF levels, which is essential for understanding its role in various physiological and pathological conditions.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (40 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (23 mentions)
Neurobasal medium, by Thermo Fisher Scientific (22 mentions)
Horse serum, by Thermo Fisher Scientific (16 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (16 mentions)
B27 supplement, by Thermo Fisher Scientific (16 mentions)
Poly d lysine, by Merck Group (13 mentions)
Fetal bovine serum (fbs), by Merck Group (13 mentions)
Glutamax, by Thermo Fisher Scientific (13 mentions)
Horse serum, by Merck Group (11 mentions)
L glutamine, by Thermo Fisher Scientific (11 mentions)
Penicillin, by Merck Group (10 mentions)
Streptomycin, by Merck Group (10 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Thermo Fisher Scientific (9 mentions)
Poly l lysine (pll), by Merck Group (9 mentions)
Laminin, by Merck Group (7 mentions)
L glutamine 200 mm, by Thermo Fisher Scientific (7 mentions)
Collagenase a, by Roche (6 mentions)
Brain derived neurotrophic factor (bdnf), by Merck Group (6 mentions)
202 protocols using nerve growth factor (ngf)
1
Neuronal Differentiation of PC12 Cells
2
Neurotrophic Factors and Metabolite Treatments
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Experiments were conducted with Palmitoylethanolamide (PEA, Tocris Bioscience, cat No. 0879-10 mg, Bristol, UK), Paclitaxel (Taxol equivalent, Invitrogen, cat No. P3456-5 mg, Schwerte, Germany), Nerve Growth Factor-2.5S from the murine submaxillary gland (NGF, Sigma Aldrich, Merck, cat No. N6009-4X 25 µg, St. Louis, MO, USA) and glial cell-derived neurotrophic factor (GDNF, Sigma-Aldrich, cat No. SRP3309-10 µg, St. Louis, MO, USA), Uridine (Uridin, Sigma-Aldrich, U3003-5 g, Darmstadt, Germany), and 5-Fluoro-2-deoxyuridine (FudR, Sigma-Aldrich, cat No. F0503-100 mg, Darmstadt, Germany). PEA and Paclitaxelwere dissolved in DMSO to obtain stock solutions of 10 mM PEA and 1 mM Paclitaxel and stored at −20 °C, while NGF and GDNF dissolved in 0.1 % Bovine Serum Albumin (BSA, Sigma-Aldrich, cat No. A7906-10 g, St. Louis, MO, USA). A total of 20 mM uridine/5-fluorodeoxyuridine (UFdU) stock solution was prepared by mixing 48.8 mg uridine and 49.2 mg 5-fluorodeoxyuridine in 10 mL distilled water, and 100 μL aliquots were prepared and frozen at −20 °C. Notably, controls contained the similar highest concentration of DMSO (0.1%) to exclude any effects on investigated parameters.
3
Sensory Neuron Culture and NGF-Withdrawal
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Sensory neurons of E15 rat or E13 mouse DRGs were cultured as described (Feinberg et al., 2010 (link)). For immunostaining, neurons were plated at a density of 50 × 103 cells per 13-mm glass slide precoated with Matrigel (BD Biosciences), and poly-d -lysine (Sigma-Aldrich) as described (Feinberg et al., 2010 (link)). For phosphoproteomics, rat neurons were plated at a density of 10 × 106 cells per 10-cm dish coated with poly-d -lysine and laminin (Corning) as described (Feinberg et al., 2010 (link)). To eliminate non-neuronal cells, the day after plating, cultures were treated with 10 ng/ml CA for 2 d in growth medium composed of basal medium Eagle, ITS supplement, 0.2% BSA, 4 mg/ml d -glucose (all from Sigma-Aldrich), GlutaMAX (Gibco), 50 ng/ml NGF (Almone Labs), and antibiotics. Between CA treatments, the cultures were grown in NB medium, containing Neurobasal Medium (Gibco), GlutaMAX, 50 ng/ml NGF, B27 supplement (Gibco), and antibiotics for 2 days. After the second CA treatment, cultures were grown in NB medium for 1 additional day. For NGF-withdrawal experiments, neurons were washed twice with factor-free culture medium and then cultured in NB medium without NGF and including anti-NGF-β (1:1,000; Sigma-Aldrich).
4
Differentiation and Oxidative Stress Analysis of PC12 Cells
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Undifferentiated PC12 cells were plated onto collagen IV-coated plates (Corning) and differentiated in culture medium containing 50 ng/ml NGF (Accurate chemicals) for 5–7 days as described20 (link),21 (link). Differentiated PC12 were pre-treated with 10 mM N-acetylcysteine (NAC) (Sigma Aldrich) before NGF withdrawal and continuously replenished every 12 hours after NGF withdrawal with anti-NGF antibody (1:5000, Sigma Aldrich) for up to 48 hours where they were harvested for Immunoblot analysis.
5
Neuroprotective Effects of Medicines on NGF-Differentiated PC12 Cells
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PC12 cells, kindly donated from Professor Lu (Department of Chinese Medicine Pharmacology, Nanjing University of Chinese Medicine) were cultured at 37°C in DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; GIBCO), 100 U/ml penicillin and 100 μg/ml streptomycin (Hyclone, J150019) under a humidified atmosphere of 95% air and 5% CO2. For cell differentiation, cells were treated with 50 ng/mL of nerve growth factor (NGF; Sigma-Aldrich, USA) for 48 h. Afterwards, NGF-differentiated cultures were pretreated with different doses of each medicine at 1, 5, 10, and 20 μmol/L for 1 h, and then expored to10 mM L-glutamate for an additional 24 h. The untreated PC cells without the treatment of L-glutamate were used as control group.
6
Myelination of Mouse DRG Neurons
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Mouse DRG neurons were isolated after dissecting the spinal cord of embryonic day 13.5 (E13.5) embryos and seeded on 12 mm glass coverslips, as described (Poitelon and Feltri 2018) . In details, after isolation, DRG were dissociated and then incubated in 0.25 % Trypsin solution (25200056, Thermofisher Scientific, USA) for 45 min at 37 °C. DRGs were mechanically dissociated and ~40000 cells were seeded in matrigel (#356234, Corning, USA), USA) coated coverslips in C-medium, composed ofMEM medium (11090081, Thermofisher Scientific, USA), 2 mM L-glutamine (25030024, Thermofisher Scientific, USA), 10 % FBS (10270098, Thermofisher Scientific), 4 mg/ml D-glucose (G5146 (Sigma-Aldrich, USA), 50 ng/ml NGF (N6009, Sigma-Aldrich, USA). After 24 h, the C-medium was replaced by NB medium:Neurobasal medium (21103049, Thermofisher Scientific, USA) 4 g/l D-glucose (G5146, (Sigma-Aldrich, USA), 2 mM L-glutamine (25030024, Thermofisher Scientific, USA), 50 ng/ml NGF (N6009, Sigma-Aldrich), and B27 supplement 1 × (50X, 17504044, Thermofisher Scientific, USA) and changed every two days. After 5 days of NB medium, myelination was induced by adding 50 µg/ml ascorbic acid (A0278, Sigma-Aldrich, USA) to the c-medium, for 12 days.
7
Inhibition of NGF Signaling Pathways
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NGF (Millipore; Burlington, MA, USA) was used at 100 ng/mL. The TrkA inhibitor, GW441756 (Selleckchem, Munich, Germany) was added (at 1 μM, final concentration) 20 minutes before NGF stimulation. The mitogen-activated kinase kinase (MEKK) inhibitor PD98059 (Alexis, San Diego, CA, USA) was added (at 10 μM, final concentration) 20 minutes before NGF stimulation. The PI-3K inhibitor, LY-294002 (Millipore; Burlington, MA, USA) was added (at 10 μM, final concentration) 30 minutes before NGF stimulation [85 ].
8
Assessing Sperm Quality with Exogenous NGF
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Immediately after collection, the sperm concentration was measured using a Thoma–Zeiss counting chamber and a light microscope (Olympus CH2, Tokyo, Japan) with a 40× magnification.
An aliquot of the semen (about 0.5 mL) was centrifuged at 700× g for 15 min to obtain seminal plasma (SP) and to quantify NGF concentration by using ELISA.
An aliquot of each semen sample derived from the different collections was diluted with a modified TALP [13 (link)] to achieve a final concentration of 107 sperm/mL. The effect of storage was evaluated at different time points (0-2-4-6 h) in semen samples supplemented at time 0 with 100 ng/mL exogenous NGF (Merck, Milan, Italy), according to the dose–response curve previously described [13 (link)], and compared to vehicle samples diluted with PBS instead of NGF. The samples were evaluated for motility, viability, and necrotic/apoptotic processes as described below. Furthermore, receptor expression was also evaluated.
An aliquot of the semen (about 0.5 mL) was centrifuged at 700× g for 15 min to obtain seminal plasma (SP) and to quantify NGF concentration by using ELISA.
An aliquot of each semen sample derived from the different collections was diluted with a modified TALP [13 (link)] to achieve a final concentration of 107 sperm/mL. The effect of storage was evaluated at different time points (0-2-4-6 h) in semen samples supplemented at time 0 with 100 ng/mL exogenous NGF (Merck, Milan, Italy), according to the dose–response curve previously described [13 (link)], and compared to vehicle samples diluted with PBS instead of NGF. The samples were evaluated for motility, viability, and necrotic/apoptotic processes as described below. Furthermore, receptor expression was also evaluated.
9
Culturing and Stimulating Superior Cervical Ganglia
10
PC12 Cell Culture and Differentiation
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PC12 cells (CRL-1721; American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 5% horse serum (HS; Invitrogen; Thermo Fisher Scientific, Inc.), 10% fetal bovine serum (FBS; Invitrogen Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Invitrogen; Thermo Fisher Scientific, Inc.), under a humidified atmosphere containing 5%/95% CO2/air at 37°C. PC12 cells underwent differentiation for 48 h at 37°C with 50 ng/ml nerve growth factor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) dissolved in DMEM containing 1% FBS, 1% HS and 100 U/ml of penicillin/streptomycin.
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