Primary human cortical neurons (ScienCell) were thawed, counted, diluted in neuronal media with Neuronal growth supplement (ScienCell), and plated at 1.2 × 104cells/well in a 96-well plate precoated with poly-L-lysine (Sigma) and laminin (Millipore). Neurons were either cultured alone, or in the presence of HL60 cells, DG75 cells (1.2 × 104cells/well) or recombinant human NGF (10 μg/ml), at 37°C and 5% CO2. After 20 h neurons were fixed with ice cold 4% PFA in PBS for 30 min, prior to immunohistochemical staining and imaging.
Neuronal growth supplement
Manufactured by ScienCell
Neuronal growth supplement is a cell culture media component designed to support the growth and differentiation of neuronal cells in vitro. It provides essential nutrients and factors to promote the healthy development of neurons.
Automatically generated - may contain errors
Lab products found in correlation
Laminin, by Merck Group (2 mentions)
Primary human cortical neurons, by ScienCell (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Neuronal medium, by ScienCell (2 mentions)
Astrocyte growth supplement, by ScienCell (2 mentions)
Astrocyte medium, by ScienCell (2 mentions)
Trypsin edta solution, by Beyotime (1 mentions)
Poly l lysine (pll), by Corning (1 mentions)
6 well plate, by Corning (1 mentions)
Matrigel basement membrane matrix, by Corning (1 mentions)
Antibiotic solution, by ScienCell (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Solarbio (1 mentions)
Earle s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Percoll, by Solarbio (1 mentions)
T25 flask, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Solarbio (1 mentions)
Human astrocyte, by ScienCell (1 mentions)
6 protocols using neuronal growth supplement
1
Neuronal Coculture and Imaging
2
Isolation and Culture of Rat Cortical Neurons
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Cortical neurons were prepared from 1-day-old newborn rats as previously described [9 (link)]. Newborn rats were decapitated, and cerebral cortexes were transferred to PBS. After the removal of the meninges and blood vessels, tissues were cut into small pieces, followed by incubation in 0.25% trypsin-EDTA solution (Beyotime) at 37°C for 20 minutes. Then LG-DMEM with 20% fetal bovine serum was added to terminate the incubation. After centrifugation at 800 rpm for 10 minutes, the cells were collected for follow-up experiments.
Cortical neurons were dispersed with a neuronal medium (Sciencell) with 1% (vol/vol) neuronal growth supplement (Sciencell), and then they were seeded at a density of 5 × 105/ml onto coverslips precoated with poly-L-lysine in 6-well plates (Corning). The medium was replaced once every 3 days, and after 7 days, the cells were fixed with 4% formaldehyde and used for the identification of neurons by immunocytochemistry.
Cortical neurons were dispersed with a neuronal medium (Sciencell) with 1% (vol/vol) neuronal growth supplement (Sciencell), and then they were seeded at a density of 5 × 105/ml onto coverslips precoated with poly-L-lysine in 6-well plates (Corning). The medium was replaced once every 3 days, and after 7 days, the cells were fixed with 4% formaldehyde and used for the identification of neurons by immunocytochemistry.
3
Examining Neuronal Responses to Prolactin
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Human primary neurons (HNs) prepared from human brains were obtained from ScienCell Research Laboratories (#1520; ScienCell, San Diego, CA, USA) and seeded onto 24-well cluster plates pretreated with Matrigel® basement membrane matrix (Corning, Corning, NY, USA). Primary neurons were maintained in neuronal medium (ScienCell) supplemented with neuronal growth supplement (ScienCell) according to the manufacturer’s instructions [34 (link)]. To investigate the morphological changes in neurons induced by PRL, 1 to 100 ng/mL PRL was dropped into the medium, and the numbers of HN neurites were determined after 24 h of culture (n = 3 for each). As a control, medium with no added PRL was used. Neuronal maturation was evaluated using the ratio of cells of each neurite number (1 to 4 and ≥5) to all cells. Subsequently, to investigate the neurophysiological effects of PRL under hypoxia, 10 ng/mL PRL and 10 ng/mL T3 were dropped into the medium, respectively, and cultured under a hypoxic condition (3% O2). The percentage of dead cells due to apoptosis under hypoxia from 10 min to 24 h was measured by TUNEL staining (n = 3 for each). The ratio of nerve cell death was defined as the number of dead nerve cells/number of all nerve cells. As a control, medium with no added PRL or T3 was used.
4
Primary Astrocyte and Neuron Isolation
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Primary astrocytes were isolated from the sheared brain tissues of rats undergoing mechanical dissociation at 6 r/min for 30 min in Earle’s balanced salt solution (Gibco, Carlsbad, CA, USA) supplemented with DNase I. After filtration using a 70-µm cell strainer, cells resuspended in 30% Percoll (Solarbio) were centrifuged at 300 × g for 30 min, followed by another centrifugation at 300 × g for 10 min. Cells resuspended in the astrocyte medium (ScienceCell, Carlsbad, CA, USA) were cultured in T25 flasks (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5 mL astrocyte medium containing 2% fetal bovine serum (Gibco) and 1% astrocyte growth supplement (ScienceCell), and 1% antibiotic solution (ScienceCell) under the condition of 37 °C and 5% CO2. Also, primary neurons were isolated from brain tissues. Sheared tissues were incubated in the mixed solution consisting of 0.25% trypsin (Solarbio) plus 1% collagenase I (Solarbio) for 30 min at 37 °C. After filtration using a 100-µm cell strainer, cells resuspended in neuronal medium (ScienceCell) were incubated in T25 flasks containing neuronal medium with 1% neuronal growth supplement (ScienceCell) and 1% antibiotic solution (ScienceCell) at 37 °C/5% CO2. In non-MOOs groups, cells were incubated with the vehicle at the equal volume to MOOs.
5
Differentiation of SH-SY5Y Cells and Astrocyte-Conditioned Medium Preparation
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Human-derived SH-SY5Y cells were cultured in Dulbecco's Modified Eagle Medium/ Ham's Nutrient Mixture F-12 (DMEM/F12K) + 10% FCS and differentiated to a neuronal phenotype by incubation in neuronal medium (ScienCell 1521) for 1 week. This medium contains Neuronal Growth Supplement (ScienCell 1562), which provides retinyl acetate (final concentration = 0.01 μg/mL) and growth factors, hormones, and antioxidants necessary for neuronal cell culture. This treatment produced cells comparable to other retinoic acid differentiation protocols such as that described by Encinas et al. [16] (link). Differentiated cells were used within 3 days. Medium could be replaced with phosphate-buffered saline (PBS) for up to 1 hour. For longer-term experiments, the continued presence of medium was necessary for cell survival. Human astrocytes (ScienCell) were cultured in Astrocyte Medium containing 2% fetal bovine serum, astrocyte growth supplement (ScienCell 1852), and penicillin/streptomycin. After expansion, astrocytes were replated in serum-free medium (DMEM/F12, 1:1) and serum-free astrocyte-conditioned medium was collected after 48 hours and stored at −20°C.
6
Neuronal Coculture and Imaging
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