All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using protocols approved by the Institutional Animal Care and Use Committee at Stanford University. Primary embryonic mouse hippocampal neuron cultures were prepared as previously described19 (link). Briefly, neurons were plated on poly-D-lysine-coated (10 µg/ml) glass cover slips at 40,000–50,000 cells per well in 12-well plates (Corning Life Sciences, Tewksbury, MA). Cells were seeded in plating medium DMEM/F12 supplemented with 10% fetal bovine serum and 1 × penicillin/streptomycin (PS) for 2 h. Medium was then changed to Neurobasal medium (Invitrogen, Carlsbad, CA) supplemented with PS and B27 (Invitrogen) and 2 mM glutamine. After 21 days under these culture conditions, neurons demonstrate an adult-like phenotype with respect to neuronal polarity, microtubule and neurofilament cytoskeleton architecture, dendritic spines and functional synapses40 (link). At 21 days cells were exposed to oligomeric Aβ at a concentration of 5 µM under conditions described in the “Results ” and Figure Legends.
Poly d lysine coated
Manufactured by Corning
Poly-D-lysine coated lab equipment is a specialized surface coating designed to enhance cell adhesion and growth. The coating provides a positively charged substrate that promotes the attachment and proliferation of various cell types. This product is intended for use in cell culture applications where improved cellular attachment is required.
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Lab products found in correlation
4 protocols using poly d lysine coated
1
Primary Mouse Hippocampal Neuron Culture
2
High-content imaging and cell immunofluorescence analysis
3
3D Cell Culture in Multi-Well Plates
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Cells were seeded on 24-well plates (standard tissue-culture treated, Sigma Aldrich, Cat. No. CLS3738-100EA, Laminin-coated, Corning, 354412 or Poly-d -Lysine-coated, Corning, 354619) in density of 10,000 cells/well and cultured in media 1–14 for 6–14 days (depending on confluence reach). All media initially used in the research are enumerated and described in Table 2 and S3. Specific number is dedicated to specific cell medium throughout the whole study. The cells were cultured in media accordingly with manufacturers’ instructions. Selected cell line-media sets were chosen as best for 3D culture based on confluence reach velocity and 3D structures morphology.
4
Culturing Primary Hippocampal Neurons from Neonatal Rats
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Primary hippocampal formation cultures were prepared from 24 hours postnatal SD rats. Briefly, pups were anesthetized with ether, and sterilized in 75% ethanol. Rat brains were quickly removed into ice-cold dissection solution. The hippocampi were stripped and cut into small pieces (each cube <1 mm 3 ). The tissue pieces were incubated with 0.125% trypsin at 37℃ for 15-20 min, and then complete culture medium was added to stop enzymatic reaction. Single-cell suspensions were obtained by mechanical dissociation using a Pasteur pipette with a fire-polished tip in complete culture medium, and then filtered through a 200 mesh nylon screen, centrifuged for 5 min at 1000 rpm. The cells at the bottom were resuspended with fresh complete culture medium and plated on poly-D-lysine coated 96-well plates and 35 mm culture dishes (Corning Inc) at a density of 5×10 5 cells/ml. Cultures were maintained in 5% CO 2 at 37℃in complete culture medium for 6 h. The culture media were then changed to serum-free B27/neurobasal medium. Afterward, half of the culture medium was replaced with fresh serum-free B27/neurobasal medium every 3 days. 7-10 days after plating, the mature cells were used for further experimental observation. The experiment was all carried out under sterile condition.
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