Poly dl ornithine hydrobromide

Manufactured by Merck Group

Poly-DL-ornithine hydrobromide is a synthetic amino acid polymer used in various laboratory applications. It is a water-soluble compound that can be utilized as a coating or substrate material for cell culture and other in vitro experiments.

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6 protocols using poly dl ornithine hydrobromide

Neurite Outgrowth Assay in LRRK2 Transgenic Mice

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Hippocampus from P0 transgenic and non-tg mice was dissected and dissociated with 0.25% Trypsin/EDTA (Gibco) for 14 min at 37°C. Cells were seeded on 96-well microplate (BD Falcon) pre-coated with 0.5 mg/ml poly-DL-ornithine hydrobromide (Sigma-Aldrich) in cell culture media consisting of Neurobasal-A (Gibco), GlutaMAX-I supplement (Gibco), B27 supplement (Gibco), ßFGF (5 ng/ml, Sigma-Aldrich) and vehicle control (DMSO) (Sigma-Aldrich) or LRRK2-IN-1 diluted in DMSO (0.1μM). Cell culture media was changed every 2 days until neurons were fixed with 3.7% PFA. At day in vitro (DIV) 3, 7 and 14 neuronal cultures were immunostained with mouse anti-ß-Tubulin, Class III antibody conjugated to Alexa Fluor 488 (1:50; BD Pharmingen) and the nuclear marker Hoechst 33342 (1:2000), both diluted in PBS. Fluorescent images were captured using the BD Pathway 855 High-Content Bioimager (BD Bioscience) at 20X magnification and a montage of 25 adjoining images (5x5) per well was obtained. Following image acquisition and using the BD AttoVision V1.6 Software (BD Bioscience) images were processed and analyzed for neurite outgrowth. Several parameters (see Table 1) were measured and then statistically analyzed with GraphPad Prism V6 (two-way ANOVA with Tukey’s post hoc test).

Garcia-Miralles M., Coomaraswamy J., Häbig K., Herzig M.C., Funk N., Gillardon F., Maisel M., Jucker M., Gasser T., Galter D, & Biskup S. (2015). No Dopamine Cell Loss or Changes in Cytoskeleton Function in Transgenic Mice Expressing Physiological Levels of Wild Type or G2019S Mutant LRRK2 and in Human Fibroblasts. PLoS ONE, 10(4), e0118947.

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Culturing spinal motoneurons for co-immunoprecipitation

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Spinal motoneurons were cultured as previously described^{46 (link)}. Briefly, lumbar spinal cord tissues from E12.5 CD-1 mouse embryos were dissected, and motoneurons were enriched via p75NTR antibody panning. Motoneurons were cultured in neurobasal medium (Gibco) supplemented with B27 (1:50; Gibco), 2% heat-inactivated horse serum (Linaris), 500 µM GlutaMAX (Gibco), and 5 ng/ml BDNF. The medium was replaced 24 h after plating and then every second day. For co-immunoprecipitation, 1.5 × 10⁶ motoneurons were plated in individual T-25 flasks precoated with poly-d/l-ornithine hydrobromide (Sigma, P8638) and laminin-111 (Invitrogen, 23017-015), and grown for 6 d. On day 6, motoneurons were treated with 1 µg/ml ActD or DMSO for 6 h prior to harvesting.

Ji C., Bader J., Ramanathan P., Hennlein L., Meissner F., Jablonka S., Mann M., Fischer U., Sendtner M, & Briese M. (2021). Interaction of 7SK with the Smn complex modulates snRNP production. Nature Communications, 12, 1278.

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Microfluidic Isolation of Axonal Transport

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Dorsal root ganglia (DRG) from the spine of new-born BALB/c mice less than 3 days were collected, washed, and digested by Collagenase/Dispase (Roche) at 37°C in the CO₂ incubator for 45–60 min. The neurons were separated from each other sufficiently, filtered, washed, and cultured in Neurobasal Medium (Gibco) supplemented with 100 ng/ml nerve growth factor 2.5S (Invitrogen), 2% B27 (Gibco), and 1% penicillin and streptomycin with 2 mM glutamine (Invitrogen). Before the neuron was planted in one side of the microfluidic chamber, the coverslips were treated with Poly-DL-ornithine hydrobromide (Sigma) for one night and laminin (invtrogen) for at least 6 h and washed with Hanks’ balanced salt solution (HBSS) buffer two times, dried completely, then covered with microfluidic device, neurons were added in one well of microfluidic device, flowed into the chamber connected with two adjacent wells, after 3 days culturing, the axons grow to another chamber along the chamber microgrooves (see illustration). Infection was performed by replacing the Neurobasal Medium in the distal wells and changed with 10⁷ TCID₅₀ rPRV SC-UL36-EGFP. Time-lapse imaging was achieved by automated sequential capture.

Qi H., Wu H., Abid M., Qiu H.J, & Sun Y. (2020). Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing. Frontiers in Microbiology, 11, 1168.

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Isolation of Mouse Dorsal Root Ganglia

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Male mice were euthanized under deep CO₂ anesthesia followed by decapitation. Thoracic and lumbar DRGs were harvested and transferred to Minimum Essential Medium Eagle solution (MEM, Gibco, #11095080). Ganglia were incubated in 0.125% collagenase type IV (Gibco, # 17104019) for 2 hours followed by incubation with 0.25% trypsin (Gibco, #25200056) for 6 min. Enzymatic dissociation steps were carried out at 36 °C. DRGs were washed three times with MEM and carefully dissociated, passing through a pipette using up and down movements (Linhart et al., 2003). A 10% BSA (Miltenyi Biotec #130-091-376) gradient was used to remove the debris from the tissue and separate a pellet of cells. The cells were resuspended and plated in glass bottom dishes coated with Poly-DL-ornithinehydrobromide (Sigma P8638) and maintained in DMEM containing penicillin and streptomycin (ThermoFisher, #15140122). Cultures were maintained in the incubator at 37°C with a 5% CO₂ atmosphere for 24 hours.

Navia-Pelaez J.M., Lemes J.B., Gonzalez L., Delay L., dos Santos Aggum Capettini L., Lu J.W., Dos Santos G.G., Gregus A.M., Dougherty P.M., Yaksh T.L, & Miller Y.I. (2022). AIBP regulates TRPV1 activation in CIPN by controlling lipid raft dynamics and proximity to TLR4 in DRG neurons. Pain, 164(6), e274-e285.

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Neural Progenitor Cell Isolation and Culture

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NPCs were isolated from embryonic day 13.5 Ai9-tdTomato homozygous mouse brains. Cells were cultured as neurospheres at 37 °C with 5% CO₂ in NPC medium: DMEM/F12 (Gibco, CAT# 10565018) with GlutaMAX supplement, sodium pyruvate, 10 mM HEPES, nonessential amino acid (Gibco, CAT# 11140076), penicillin and streptomycin (Gibco, CAT# 10378016), 2-mercaptoethanol (Gibco, CAT# 21985023), B-27 without vitamin A (Gibco, CAT# 12587010), N2 supplement (Gibco, CAT# 17502048), and growth factors, bFGF (BioLegand, CAT# 579606) and EGF (Gibco, CAT# PHG0311) (both 20 ng/ml as final concentration). NPCs were passaged using MACS Neural Dissociation Kit (Papain, CAT# 130-092-628) following manufacturer’s protocol. bFGF and EGF were refreshed every three days and cells were passaged every 5 days. Pre-coating with a coating solution containing poly-DL-ornithine hydrobromide (Sigma-Aldrich, CAT# P8638), laminin (Sigma-Aldrich, CAT# 11243217001), fibronectin bovine plasma (Sigma-Aldrich, CAT# F4759) was required for culturing cells in 96-well plates.

Chen K., Stahl E.C., Kang M.H., Xu B., Allen R., Trinidad M, & Doudna J.A. (2024). Engineering self-deliverable ribonucleoproteins for genome editing in the brain. Nature Communications, 15, 1727.

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Primary Hippocampal Cell Culture Protocol

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Primary hippocampal cell cultures were prepared from E16 ICR mice. The embryos were removed and decapitated, and the entire hippocampus was dissected under sterile conditions. After enzymatic digestion for 5 minutes by 0.25% trypsin at 37 C, the cells were separated by trituration in Dulbecco's modified Eagle's medium (Wako; Osaka, Japan) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific; Waltham, MA) and 1% penicillin-streptomycin (Thermo Fisher Scientific; Waltham, MA). After trituration, the solution was centrifuged at 190g for 3 minutes, and the cell pellet was immediately resuspended in Neurobasal medium (Thermo Fisher Scientific; Waltham, MA) with 2% B27 (Invitrogen; Waltham, MA), 2 mM L-gluta- mine (Nacalai Tesque; Kyoto, Japan), and 1% penicillin-streptomycin (Thermo Fisher Scientific; Waltham, MA). The dissociated cells were plated on 24-well plates (1.5 × 10 5 cells/well) that were precoated with poly-DL-ornithine hydrobromide (Sigma-Aldrich; St. Louis, MO). Half the medium was removed and replaced every 3-4 days. The cells were cultured under constant conditions of 37 C, 5% CO 2 in a humidified incubator. The experiments were conducted over 14-17 days in vitro (DIV), and each experiment was repeated 3 times.

Ueda J., Uemura N., Sawamura M., Taguchi T., Ikuno M., Kaji S., Taruno Y., Matsuzawa S., Yamakado H, & Takahashi R. (2021). Perampanel Inhibits α-Synuclein Transmission in Parkinson's Disease Models. Movement disorders : official journal of the Movement Disorder Society, 36(7).

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