Primary neuronal cultures were prepared from the hippocampi of embryonic day-15 WT and Tg2576 mice as described by Ciccone et al. [13 (link)].The cells were plated on 35 mm culture dishes coated with poly(D)-lysine hydrobromide with a molecular weight >300,000 (Sigma-Aldrich, Milan, Italy) or onto 25 mm glass coverslips (Glaswarenfabrik Karl Hecht KG, Sondheim, Germany), coated with 100 μg/mL poly(D)-lysine hydrobromide with a molecular weight of 30,000–70,000 (Sigma-Aldrich, Milan, Italy) at a density of one embryo hippocampus/1 mL. Cytosine β-D-arabinofuranoside (10 μM, Sigma-Aldrich, Milan, Italy) was added 3 days after plating to inhibit non-neuronal cell growth. The neurons were cultured at 37 °C in a humidified 5% CO2 atmosphere. The experiments were performed between 10–12 days in vitro (DIV).
Poly d lysine hydrobromide
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
Poly-D-lysine hydrobromide is a synthetic amino acid polymer commonly used as a coating for cell culture surfaces. It enhances cell adhesion and promotes cell growth in in vitro applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (18 mentions)
Neurobasal medium, by Thermo Fisher Scientific (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Glutamax, by Thermo Fisher Scientific (9 mentions)
Laminin, by Merck Group (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Triton x 100, by Merck Group (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Glutamax 1, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Merck Group (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (5 mentions)
B27 supplement, by Thermo Fisher Scientific (5 mentions)
Papain, by Worthington (4 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Phosphate buffered saline (pbs), by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
113 protocols using poly d lysine hydrobromide
1
Culturing Hippocampal Neurons from WT and Tg2576 Mice
2
STR Profiling for Cell Line Authentication
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Short tandem repeat (STR) profiling for cell line authentication of HEK 293T/17, Y-79, and WERI-RB-1 cell lines was achieved before performing the luciferase assays (Eurofins Genomics, Wolverhampton, UK). Corning white costar 96-well plates (Corning Optical Communications, Flintshire, UK) were coated with 0.2 mg/ml of poly-D-lysine hydrobromide (Merck Life Science UK Limited, Gillingham, UK) for 5 min at 37 °C, and the wells were rinsed with water before applying 0.05 mg/ml of human fibronectin (Merck Life Science) and incubating for 30 min at room temperature. After removal of the human fibronectin solution, plates were air dried for 2 h. HEK 293T/17 cells were seeded at 2E+05 cells/ml in no phenol red high glucose DMEM, pyruvate and FBS purchased from Life Technologies Ltd, Paisley, UK, L-glutamine and pen&strep purchased from Merck Life Science, 10% fetal bovine serum (FBS), and 1% penicillin and streptomycin. Y-79 and WERI-RB-1 cells were seeded at 5E+05 cells/ml in RPMI 1640 (Life Technologies Ltd, Paisley, UK) 1640 with HEPES and NHCO3 supplemented with 1% L-glutamine, 1% penicillin and streptomycin, and 10% FBS. A transfection mix of Opti-MEM (Life Technologies Ltd), ViaFect Transfection Reagent (Promega), and 400 ng of plasmid was prepared per well and applied immediately after cell seeding. Samples were incubated for 72 h at 37 °C and 5% CO2.
3
Differentiation of iPSC-derived Neurons
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iPSC-derived neurons were obtained as previously described [33 (link)], with some modifications. Briefly, control and ULD-hiPSCs were induced toward neural stem cells (NSCs) differentiation using Gibco® PSC Neural Induction Medium (Thermofisher Scientific, Waltham, MA, USA), according to manufacturer’s instructions. For the neuronal differentiation, NSCs were seeded at the density of 5 × 106 on 10-mm dishes pre-treated with 1× Poly-D-lysine hydrobromide having a molecular weight of 30,000–70,000 (Merck, Darmstadt, Germany) and 5 μg/mL Laminin (Sigma-Aldrich, St. Louis, MO, USA). Neurons were cultured in Neuronal Differentiation Medium (NDMC) composed of Neurobasal Medium, 1× B27™, 1× GlutaMAX™, 2× CultureOne™ Supplement, 200 μM L-ascorbic acid and 0.2% Penicillin/Streptomycin (all from Thermo Fisher Scientific, Waltham, MA, USA). NDMC was completed with GDNF (10 ng/mL) and BDNF (20 ng/mL) (both from PeproTech, London, UK). After 6 days, cytokine concentration was reduced to 5 ng/mL for GDNF and to 10 ng/mL for BDNF. On day 15 of differentiation, GDNF was removed while BDNF was kept at 10 ng/mL. Neurons were maintained for 21 days in a humidified incubator at 37 °C at 5% CO2 and differentiation medium refreshed every three days. At day 21 of differentiation, neurons were harvested and processed for characterization (Figure S3 ) and for molecular validation of genetic data.
4
Immunofluorescence Staining Protocol
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Cells were cultured on coverslips coated with Poly-D-lysine hydrobromide (#P7280, Sigma-Aldrich Inc., MO) were rinsed with PBS, fixed with 4 % paraformaldehyde in PBS for 30 min, and permeabilized using 0.5% Triton X-100 with 200 mM glycine for 10 min. Fixed cells were blocked with 3% BSA-containing PBS for 1 h at room temperature, and incubated with primary antibody overnight at 4 °C. Then, incubated with alexa-labeled secondary antibodies (Goat anti-Rabbit IgG Alexa Fluor 488, #A11008, Thermo Fisher Scientific, CO) for 1.5 h at room temperature. Cells were stained with DAPI (#GTX16262, GeneTex, CA) for 10 min at room temperature and mounted. Images were acquired with Zeiss LSM 880 with Airyscan (Carl Zeiss, NY).
5
Fluorescent Compound Labeling Assay
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For cell labelling studies, cells were plated onto 96-well plates (Cell Carrier; Perkin-Elmer, Waltham, MA, USA) pre-treated with poly-D-lysine hydrobromide 0.1 mg/mL (Sigma Aldrich, Spain) at a density of 10000 cells/well and cultured for 24 h. For cell labelling with compound 12i , cultured media was removed and cells were washed with HBSS supplemented with 0.1% BSA and incubated in the same buffer for 30 min at 37 °C. The buffer was removed and cells were incubated in the absence or presence of the compound at the indicated concentrations in HBSS for 10 min at 37 °C. A 10−2 M stock solution of the compound was prepared in DMSO and then diluted to the final assay concentration in HBSS. Hoechst 33342 (1 µg/mL) (Thermo Fisher, Spain) was used to stain nuclei where indicated. After the incubation time, the supernatant was removed, cells were washed twice with HBSS and plates were subjected to microscopy. Fluorescence images were acquired using an Operetta high content imaging instrument (Perkin-Elmer, Waltham, MA, USA) at 20x and 40x magnification, at excitation wavelengths of 520–550 nm and emission wavelengths of 560–630 nm (standard 5-TAMRA filter set) for compound 12i , and excitation wavelengths of 360–400 nm and emission wavelengths of 410–480 nm for Hoechst 33342 (standard Hoechst 33342 filter set).
6
Isolation and Culture of Rat Hippocampal Neurons
7
Primary Cell Culture Isolation
8
Neuronal Culture Reagents and Protocols
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Trypsin, penicillin, streptomycin, heat-inactivated fetal bovine serum, horse serum, and soybean Trypsin inhibitor were obtained from Atlanta Biologicals (Norcross, GA, USA). Minimum essential medium (MEM), deoxyribonuclease (DNase), poly-L-lysine, poly-D-lysine hydrobromide, cytosine arabinoside, NMDA, protease inhibitor cocktail, MK-801, and ifenprodil were purchased from Sigma (St. Louis, MO, USA). Pluronic acid and Fluo-3 AM were purchased from Molecular Probes (Eugene, OR, USA). TCN-201, IPA-3, and NSC23766 were purchased from Tocris (Bristol, UK). Pierce ECL kits (Thermo Fisher Scientific, Rockford, IL, USA), Neurobasal, and B-27 supplements were purchased from Invitrogen Corporation (Carlsbad, CA, USA); the p-PAK1 antibody (Thr 212) from Santa Cruz Biotechnology (Dallas, TX, USA); and the PAK1 antibody anti-rabbit IgG HRP-linked antibody from Cell Signaling Technology (Danvers, MA, USA). Brevetoxin-2 (PbTx-2) was isolated and purified from Karinia breve cultures at the Center for Marine Sciences at the University of North Carolina (Wilmington, NC, USA). QNZ-46 was a gift from SF Traynelis, Department of Pharmacology, Emory University, Atlanta, GA. The GluN2D subtype of NMDA receptor knockout mice was obtained from Daniel T. Monaghan, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska.
9
Astrocyte-enriched Cultures from Mice Forebrain
10
Primary Rat Hippocampal Neuron Culture
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Cell suspensions were prepared from rat hippocampus as described previously [21 (link)]. Coverslips (for immunofluorescence staining and calcium imaging) and culture dishes (for any other application) were coated with poly-D-lysine hydrobromide (Sigma-Aldrich, Taufkirchen, Germany). Cell suspension was preplated onto an uncoated flask and incubated at 37 °C in 5% CO2 for 1 h. During this time glial cells settled down and adhered to the bottom of the flask, while neurons remained in the supernatant. Supernatant was collected thereafter and centrifuged at 800 rpm for 8 min at room temperature. Cell pellets were resuspended and cultured in serum-free Neurobasal-A medium supplemented with 2% B27, 0.5 mM GlutaMAX and 1% penicillin-streptomycin (all Life Technologies, Darmstadt, Germany). Cells were plated on poly-D-lysine coated dishes or coverslips at a density of 2.5 × 105 onto 3.5 cm2. Cells were maintained at 37 °C in a fully humidified incubator containing 5% CO2. After 24 h Cytosine β-D-arabinofuranoside hydrochloride (AraC; Sigma-Aldrich) was added to inhibit proliferation of remaining glial cells. Neurons were maintained in dispersed culture with the original media up to 40 days in vitro (DIV).
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