Poly d ornithine

Manufactured by Merck Group

Sourced in United States

Poly-d-ornithine is a synthetic polymer composed of the amino acid D-ornithine. It is commonly used as a cell culture substrate to promote cell adhesion and growth. Poly-d-ornithine serves as a positively charged surface coating that enhances the attachment of various cell types in in vitro experiments.

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7 protocols using poly d ornithine

Transient Transfection of NG108-15 Cells

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NG108-15 cells were transiently transfected with 300 ng of each cDNA expression plasmids (see Supplementary file 1) by using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to its handbook. Briefly, the plasmids (Supplementary file 1) and the Lipo2000 were diluted into Opti-MEM Reduced Serum Medium (Gibco), respectively. Five minutes later, we added the diluted DNA to the diluted Lipo2000 to make the plasmid DNA-lipid complexes. After incubating 30 min, we plated the cells on the 12 mm round coverslips coated with ×1 Poly-D-Ornithine (Sigma-Aldrich) in 12-well plate, and in the meanwhile, we added DNA-lipid complexes to the cells. We performed membrane isolation and confocal experiments about 56 hr after transfection.

Galvanetto N., Ye Z., Marchesi A., Mortal S., Maity S., Laio A, & Torre V. (2022). Unfolding and identification of membrane proteins in situ. eLife, 11, e77427.

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Neuronal Differentiation of Ehmt1 Mutant mESCs

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Analysis used two independent clonal populations of mESC Ehmt1^+/− cell lines mutant mouse ES cells (mESCs), obtained from the European Mouse Mutant Cell Repository Centre (EuMMCR); each had a single copy of a ‘knockout first’ conditional allele Ehmt1^{tm1a(EUcOMM)Hmgu} allele [32 (link)]. Control (Ehmt^flp) a cell lines were generated using the flp-allele to restore a wild type gene. mESCs were grown on gelatin-coated plates in knockout DMEM (Gibco), supplemented with ESC certified FBS (Invitrogen), L-Glutamine (Gibco), 2-mercaptoethanol (Sigma) and ESGRO leukaemia inhibitory factor (LIF) (Chemicon) at 37 °C. Feeder-free neuronal differentiation was initiated in media lacking LIF for 4 days and 5 μM Retinoic acid was added to the culture until day 8. At this stage, cells were dissociated with 0.05% trypsin (Sigma) and seeded at 1.5 ×10⁵ per cm² density on Poly‐D-ornithine (Sigma)/laminin (Roch) in N2 medium (Sigma) [33 (link)].

Alsaqati M., Davis B.A., Wood J., Jones M.M., Jones L., Westwood A., Petter O., Isles A.R., Linden D., Van den Bree M., Owen M., Hall J, & Harwood A.J. (2022). NRSF/REST lies at the intersection between epigenetic regulation, miRNA-mediated gene control and neurodevelopmental pathways associated with Intellectual disability (ID) and Schizophrenia. Translational Psychiatry, 12, 438.

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Motor Neuron Culture and Transfection

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Glass coverslips were coated with poly-d-ornithine and laminin (15 μg/ml and 1 mg/ml, respectively; Sigma). Motor neuron cultures were performed as previously described (Murray et al., 2010 (link)). For preparation of spinal motor neurons, we used the ventral portion of the cervical and thoracic spinal cord, which is enriched in motor neurons at HH stages 28–29 (E6). Dissociated neurons were plated on laminin-coated coverslips, in Neurobasal medium containing 2% (v/v) B27 supplement, 2% (v/v) horse serum, 0.1% (v/v) β-mercaptoethanol, 0.35% (v/v) Glutamax, 1% (v/v) penicillin/streptomycin (all from Gibco), 1% (v/v) chick embryo extract and 50 ng/ml CNTF (R&D Systems). Neurons were transfected with EGFP-WT, EGFP-Q, EGFP-M or pEGFP-C1 construct as a control, using the chick nucleofection kit and an Amaxa machine (Lonza). The efficient transfection of these constructs was determined by immunostaining using anti-GFP antibodies.

Tripathi V.B., Baskaran P., Shaw C.E, & Guthrie S. (2014). Tar DNA-binding protein-43 (TDP-43) regulates axon growth in vitro and in vivo. Neurobiology of Disease, 65(100), 25-34.

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Differentiation of hESCs into NCLCs

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Use of human embryonic stem cells (HUES-7 line; Harvard University, Cambridge, MA, USA) was approved by the German Central Ethics Committee for Stem Cell Research (project codes: #1710-79-1-4-51 and 1710-79-1-4-51-E01). A quantity of hESCs were cultured on Matrigel (Corning)-coated dishes in E8-medium (self-made according to Chen et al., 2011) at 37 °C and 5% CO₂ until 80% confluency. Cells were detached using EDTA (0.5 mM) and transferred onto uncoated dishes in sphere medium (DMEM:F12, Neurobasal, N2-supplement, B27-supplement, glutamine (all ThermoFisher Scientific, Waltham, MA, USA)), insulin (CSBio) with freshly added EGF and FGF (20 ng/μL; Peprotech). ROCK inhibitor (10 μM; Stem Cell Technologies) was added for up to 48 h, depending on size of spheres. One day after plating, small spheres become visible. The medium was changed every other day. After 7–9 days, spheres spontaneously attach and NCLCs start to migrate out. NCLCs were collected using Accutase (Sigma) after remnants of spheres were manually removed from the plate. NCLCs can be frozen in liquid nitrogen or immediately plated on Poly-D-ornithine (15 μg/μL, Sigma), laminin (10 μg/mL, Sigma) and fibronectin (10 μg/mL, ThermoFisher Scientific)-coated 3.5 cm dishes in a density of 60,000 cells/cm² in sphere medium without FGF and EGF.

Schrenk-Siemens K., Pohle J., Rostock C., Abd El Hay M., Lam R.M., Szczot M., Lu S., Chesler A.T, & Siemens J. (2022). Human Stem Cell-Derived TRPV1-Positive Sensory Neurons: A New Tool to Study Mechanisms of Sensitization. Cells, 11(18), 2905.

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Isolation and Culture of Cortical Neural Stem Cells

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Cortical NSCs were obtained from pregnant imprinting control region mice (E13.5). The cortices were dissected and washed 1–2 times with Hank's Balanced Salt Solution (HBSS; HyClone Laboratories, South Logan, UT, USA). To each piece of washed tissue, 5 mL of HBSS were added, and the tissue was dissociated by pipetting up and down. Tissues were triturated by repeated passages through a fire-polished constricted Pasteur pipette. Dissociated tissues were allowed to settle for 3 min. Supernatants were transferred to a fresh tube, and were centrifuged at 1200 g for 3 min. Pellets were resuspended in NSC basal media with a proliferation supplement (Stem Cell Technologies, Vancouver, Canada), and 20 ng/mL epidermal growth factor (EGF, Invitrogen, Carlsbad, CA, USA). NSCs were plated on poly-D-ornithine (Sigma-Aldrich, St. Louis, MO, USA) treated dishes at a density of 2.5×10⁴ cells/mL. Cultures were maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37℃. Culture medium was replaced every 3 days. NSCs were used for experiments after 2–3 passages.

Song J., Oh Y., Kim J.Y., Cho K.J, & Lee J.E. (2016). Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation. Yonsei Medical Journal, 57(6), 1461-1467.

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Embryonic Midbrain Neuron Culture

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Primary DA neuron cultures were prepared from embryonic ventral midbrain using microisland methods according to published procedures (Takeshima et al., 1996 (link); Tang et al., 2010 (link)). Briefly, E13.5 mouse embryos were collected from timed-pregnant females. The ventral midbrain was dissected, dissociated after treatment with trypsin and cultured on coverslips coated with poly-D-ornithine (Sigma) and laminin (Sigma) at the density of 1.0 × 10⁶/mL. The dissociated cells were maintained in DMEM/F-12 (1:1) medium containing 10% FBS overnight. Then, the media was changed to DMEM/F-12 (1:1) medium containing N2 supplements (Invitrogen), 20 ng/mL FGF2 (Millipore Corporation), 100 ng/mL FGF8 (PeproTech), and designated factors, including 1–10 ng/mL TGF-b1 (R&D), TGF-β3 (R&D), and 1 μM SB-431542 (Sigma) for additional 2 days in vitro (DIV) before they were fixed and processed for immunofluorescence. For each treatment condition, 30 neurons from three separate embryonic brain cultures were analyzed.

Luo S.X., Timbang L., Kim J.I., Shang Y., Sandoval K., Tang A.A., Whistler J.L., Ding J.B, & Huang E.J. (2016). TGF-β Signaling in Dopaminergic Neurons Regulates Dendritic Growth, Excitatory-Inhibitory Synaptic Balance, and Reversal Learning. Cell reports, 17(12), 3233-3245.

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Neurochemical Signaling Pathways: Protocols

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5-Hydroxytryptamine/5-HT (Sigma Aldrich, H9523), 2,5-dimethoxy-4-iodoamphetamine/DOI (Sigma Aldrich, D101), 3,4-dihydroxyphenethylamine/dopamine or DA (Sigma Aldrich-H8502), chlorpromazine (Sigma, 8138), G418 and poly-d-ornithine were purchased from Sigma-Aldrich, USA. Risperidone (Tocris, 2865), olanzapine (Tocris, 4349), phorbol 12-myristate 13-acetate/PMA (Tocris, 1201) and A23187 (Tocris 52665-69-7) were purchased from Tocris, USA. Calphostin C (Cayman, 121263-19-2), CCG-1423 (Cayman 285986-88-1), SU6656, W7 (Cayman, 61714-27-0), latrunculin-A (Cayman, 76343-93-6), were purchased from Cayman Chemical, USA. Fibronectin, PrestoBlue, Phalloidin Alexa-Fluor 568 were purchased from Invitrogen, USA.

John Jayakumar J.A., Panicker M.M, & Basu B. (2020). Serotonin 2A (5-HT2A) receptor affects cell–matrix adhesion and the formation and maintenance of stress fibers in HEK293 cells. Scientific Reports, 10, 21675.

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