Hippocampal neurons from male and female ICR E16 (Charles River Laboratories, Japan) or male and female Wistar rat E17‐18 (Charles River Laboratories) were cultured on 0.01% poly‐L‐ornithine (Wako Pure Chemicals, Japan)‐coated dishes in neurobasal medium (Gibco, U.S.A) supplemented with 2% B27 supplement (Gibco, U.S.A) and 0.5mM L‐glutamine (Nacalai Tesque, Japan). Neurons were cultured for 21–28 days in vitro (DIV) on plastic coverslips.
Poly l ornithine
Manufactured by Fujifilm
Sourced in Japan
Poly-L-ornithine is a synthetic polymer composed of the amino acid L-ornithine. It is commonly used as a cell culture surface coating agent to promote cell adhesion and growth. The polymer facilitates the attachment and proliferation of various cell types, making it a valuable tool in cell biology research and tissue engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (5 mentions)
Macs neuro medium, by Miltenyi Biotec (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Nacalai Tesque (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Neurobrew b21, by Miltenyi Biotec (1 mentions)
Papain dissociation system, by Worthington (1 mentions)
Recombinant human basic fibroblast growth factor, by Fujifilm (1 mentions)
Fibronectin, by Fujifilm (1 mentions)
Ascorbic acid, by Fujifilm (1 mentions)
B27 supplement, by Fujifilm (1 mentions)
Y 27632, by Fujifilm (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Dibutyryl camp, by Santa Cruz Biotechnology (1 mentions)
Neurobasal plus medium, by Thermo Fisher Scientific (1 mentions)
Laminin, by Merck Group (1 mentions)
Stemdiff neural rosette selection reagent, by STEMCELL (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
6 protocols using poly l ornithine
1
Culturing Hippocampal Neurons from Rodents
2
Neurodegenerative Disease Modeling in Microfluidics
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A glass-bottom dish with 30 mm glass diameter (Mat Tek Corporation, P50G-0-30-F) was coated with 0.2 mg/mL poly-L-ornithine (FUJIFILM Wako Pure Chemical Corporation, 27378-49-0) for 1 h at 37 °C, washed with autoclaved water, and a microfluidics device was attached (Xona Microfluidics LCC, SND450-1). Primary cortical neurons were collected from E15.5 C57BL/6 J mouse pups, dissociated using a papain dissociation system (Worthington Biochemical Corporation, LK003150), and cultured on both sides of the microfluidics chamber in MACS Neuro Medium (Miltenyi Biotec, 130-093-570) supplemented with NeuroBrew B21 (Miltenyi Biotec, 130-097-263), 1% GlutaMAX (Thermo Fisher Scientific, 35050-061), and 1% penicillin and streptomycin. At 3 days in vitro (DIV), the glial inhibitor 5-fluoro-2-deoxyuridine was added. The donor neurons were treated with 10 µg/mL PFF550 at DIV7, and the recipient neurons were treated with 100 nM bafilomycin A1 and/or 1 mM LLOMe for 1 h at DIV11 and fixed by 4% paraformaldehyde at DIV18.
Further details of the experimental procedure regarding antibodies and reagents, plasmids, cell culture, western blotting, siRNA knockdown, immunofluorescence and microscopy observation, live-cell imaging, and TEM are available inSI Appendix, Materials and Methods .
Further details of the experimental procedure regarding antibodies and reagents, plasmids, cell culture, western blotting, siRNA knockdown, immunofluorescence and microscopy observation, live-cell imaging, and TEM are available in
3
Neural Stem Cell Differentiation from iPSCs
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NSph, neural stem cell derived from human iPSC, were cultured as previously described (Matsumoto et al., 2016) . Briefly, iPSCs were dissociated into single cells with TrypLE Select (Thermo Fisher Scientific), and then cultured in Neurobasal Plus Medium (Thermo Fisher Scientific) supplemented with B27 supplement (Thermo Fisher Scientific), 20 ng/mL recombinant human basic fibroblast growth factor, 10 µM Y-27632 (Fujifilm Wako Pure Chemical), and 10 ng/mL human leukemia inhibitory factor (Fujifilm Wako Pure Chemical) under 5% oxygen. NSphs were repeatedly passaged by dissociation into single cells and then cultured as described above. NSph diameter was measured using the ImageJ software provided by the National Institute of Health (http://rsb.info.nih. gov/ij/). For neural differentiation, NSphs were adhered to poly-L-ornithine (Fujifilm Wako Pure Chemical)-and fibronectin (Fujifilm Wako Pure Chemical)-coated dishes and cultured in Neurobasal Plus Medium supplemented with B27 supplement, 10 ng/mL brain-derived neurotrophic factor (Fujifilm Wako Pure Chemical), 10 ng/mL glial cell-derived neurotrophic factor, 1 mM dibutyryl-cAMP (Santa Cruz Biotechnology, CA, USA), and 200 µM ascorbic acid (Fujifilm Wako Pure Chemical) for 14 days. All experiments were independently performed at least twice.
4
Derivation of Human Long-Term Neuroepithelial Stem Cells
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We generated human lt-NES cells from 201B7 and P11025 cells according to the methods of Falk et al. 15 The generation procedures were as follows: 1.5 × 10 6 cells were plated in Aggrewell 800 plates (STEMCELL Technologies, Vancouver, Canada) in STEMdiff Neural Induction medium (STEMCELL Technologies). Neural aggregates were harvested on day 5 and cultured in wells coated with poly-l-ornithine (Sigma-Aldrich) and laminin (Sigma-Aldrich). On day 12, neural rosettes were isolated by using STEMdiff Neural Rosette selection reagent (STEMCELL Technologies) and cultured in suspension for 3 days. Subsequently, the neural aggregates were transferred to poly-l-ornithine/laminin-coated dishes and maintained in lt-NES medium (DMEM/F12 [Wako] supplemented with glucose [26.5 mM; Sigma-Aldrich], 1× N2 supplement [Thermo Fisher Scientific], 0.05× B27 supplement [Thermo Fisher Scientific], 1× penicillin/streptomycin [Thermo Fisher Scientific], 10 ng/mL basic fibroblast growth factor [PeproTech, Rocky Hill, NJ], and 10 ng/mL epidermal growth factor [PeproTech]) at 37 °C in a humidified atmosphere of 20% O 2 . The medium was changed every 2 days.
5
Visualizing HMGA2 Binding to Polynucleosomes
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Polynucleosomes (150 nM) and recombinant HMGA2 (1 μM) were incubated together for 10 min at 37 °C in 100 µl of a solution containing 28 mM Tris-HCl (pH 7.5−8.0), 20 mM NaCl, 3% glycerol, 1.2 mM dithiothreitol, 0.4 mM β-mercaptoethanol, and bovine serum albumin (BSA, 5 µg/ml), after which glutaraldehyde was added to a final concentration of 0.1%. The mixture was maintained for 30 min on ice, dialyzed overnight at 4 °C with a dialysis buffer consisting of 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, and 15 mM NaCl, and stored at 4 °C. The mica substrate (Alliance Biosystems) for AFM was coated with 0.1% poly-L-ornithine (Fujifilm) for 2 min at room temperature and then washed twice with deionized water before the addition of the polynucleosomes (~6 nM) and incubation for 5 min at room temperature. The substrate was then washed twice with the dialysis buffer and observed by AFM with a NanoWizard IIR instrument (JPK) and BL-AC40TS-C2 Bio Lever Mini cantilever (Olympus). Images were acquired in QI mode with the sample in dialysis buffer. ImageJ (NIH) was used for image analysis.
6
Culturing Cortical Neurons from Pregnant Mice
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Pregnant C57BL/6J mice, (RRID:IMSR_JAX:000664) were obtained from Charles River Laboratories Japan (Yokohama, Japan). Primary cortical neurons were prepared from E15.5 pups and cultured in MACS Neuro Medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with MACS NeuroBrew-21 (Miltenyi Biotec), 0.5 mM l -glutamine, penicillin, and streptomycin (all from Invitrogen) on tissue culture plates coated with poly-l -ornithine (Fujifilm, Tokyo, Japan) at a density of 5 × 104 cells/cm2. At 3 days in vitro (DIV), a half volume of fresh media containing 2′-deoxy-5-fluorouridine (final concentration, 3.3 µM) (Tokyo Chemical Industry, Tokyo, Japan) was added to inhibit glial proliferation. At 7 DIV, half of the media was changed, and αSyn PFF (final concentration, 10 µg/mL) with or without TA was added. αSyn PFF were incubated for 7 days and then analyzed by immunocytochemistry.
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