3 x 104 cells were allowed to settle on poly-L-lysine coated slides (Menzel-Glaeser, Thermo Scientific) for 30-60min at 37°C. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 5min and subsequently permeabilized (0.1% Triton and 0.2% BSA in PBS) for 5min. Cells were then blocked in 1% BSA in PBS for 1h at RT prior to staining with the primary antibody at 4°C o/n. Cells were washed 3 times in 0.1% BSA in PBS prior to staining with the secondary antibody for 1h at RT. Cells were washed 3 times in 0.1% BSA in PBS prior to mounting in ProLong® Gold antifade reagent with DAPI (Invitrogen). Slides were sealed with nailpolish and stored at 4°C.
Poly l lysine coated
Manufactured by Thermo Fisher Scientific
Sourced in United States
Poly-L-lysine coated is a surface treatment for laboratory equipment. It is designed to enhance cell attachment to surfaces.
Automatically generated - may contain errors
Lab products found in correlation
Glass coverslip, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
24 well plate, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Bafilomycin a1 bafa1, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Trehalose, by Merck Group (1 mentions)
Neurobasal a, by Thermo Fisher Scientific (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Su8 3035 negative photoresist, by MicroChem (1 mentions)
Ca2 mg2 free hbss, by Thermo Fisher Scientific (1 mentions)
5 protocols using poly l lysine coated
1
Immunofluorescence Staining Protocol
2
Neuron-Specific Expression of STAND Proteins
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HEK293T, HeLa-S3, COS-7, and highly malignant MIA PaCa-2 pancreatic adenocarcinoma cells obtained from RIKEN Bioresource Center Cell Bank (Tsukuba, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, Osaka, Japan; #044-29765) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). Primary dopaminergic neuron cultures were prepared from the ventral mesencephalon of embryonic day 13–14 male and female mouse embryos. Briefly, the ventral mesencephalon was dissociated by treatment with trypsin (0.25% for 20 min at 37 °C; Gibco), followed by trituration with a fire-polished Pasteur pipette in neurobasal medium (Gibco) supplemented with 10% FBS containing deoxyribonuclease I (DNase I). Dissociated cells (6 × 104) were seeded on poly-l -lysine-coated (1 μg/ml) glass coverslips (diameter: 12 mm; Thermo Fisher Scientific, Waltham, MA, USA) in a 24-well plate (Falcon; Corning, NY, USA) and cultured in 500 μl of neurobasal medium supplemented with B27 (Invitrogen, Carlsbad, CA, USA). At 7 days in vitro, cells in each well were infected with 5 μl AAV vector (titre: 1 × 1011 vg/ml) to express STAND proteins in a neuron-specific manner by a synapsin promoter and analysed by immunocytochemistry or used for the DA release assay 7 days later.
3
Cortical Neuron Culture and Autophagy Assays
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Pups were decapitated, and the cerebral cortex was removed and enzymatically dissociated. Cortical neurons were plated on poly‐L‐lysine‐coated (0.1 mg/ml) glass coverslips (Thermo‐Fischer Scientific) at a density of 40,000 cells/mL and maintained up to 14 days in vitro (DIV) at 37°C, 5% CO2, 95% humidity in a culture medium consisting of Neurobasal A, B‐27 (2%), GlutaMAX (1%), and penicillin/streptomycin (1%). All chemicals were purchased from Life Technologies/Thermo‐Fischer Scientific unless stated otherwise.
For the experiments aimed at studying the autophagic flux, 30 nM rapamycin (LC Laboratories, 72 h), 300 nM Bafilomycin A1 (BafA1, 8 h), 100 mM trehalose (72 h), 2 mM 3‐methyladenine (3‐MA, 72 h) (Sigma‐Aldrich, Milano, Italy) treatments were carried. As a control for both treatments, cultures were subjected to the same medium change with addition of an equivalent volume of vehicle.
The production of VSV‐pseudotyped third‐generation lentiviral particles was performed as previously described (Rocchi et al., 2019 (link)). pLenti‐PGK‐Cre‐EGFP or pLenti‐PGK‐ΔCre‐ EGFP plasmids were obtained as previously described (Kaeser et al., 2011 (link)). Primary neurons were infected at 7 DIV at a multiplicity of infection of 10. After 24 h, half of the medium was replaced with fresh medium.
For the experiments aimed at studying the autophagic flux, 30 nM rapamycin (LC Laboratories, 72 h), 300 nM Bafilomycin A1 (BafA1, 8 h), 100 mM trehalose (72 h), 2 mM 3‐methyladenine (3‐MA, 72 h) (Sigma‐Aldrich, Milano, Italy) treatments were carried. As a control for both treatments, cultures were subjected to the same medium change with addition of an equivalent volume of vehicle.
The production of VSV‐pseudotyped third‐generation lentiviral particles was performed as previously described (Rocchi et al., 2019 (link)). pLenti‐PGK‐Cre‐EGFP or pLenti‐PGK‐ΔCre‐ EGFP plasmids were obtained as previously described (Kaeser et al., 2011 (link)). Primary neurons were infected at 7 DIV at a multiplicity of infection of 10. After 24 h, half of the medium was replaced with fresh medium.
4
Soft Lithography for Microchip Fabrication
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The microchip fabrication was performed according to standard soft lithography and microfabrication methods using SU8-3035 negative photoresist (Microchem) and polydimethylsiloxane (RTV 615, Momentive). Briefly, a 10:1 mixture of PDMS prepolymer A and cross-linker B was poured on an SU8 mold and cured in an oven at 80 °C for 1 h after vacuum degassing. The PDMS microchip was punched and bonded with a premium-grade microarray glass slide (poly-L-lysine–coated; Thermo Fisher). The whole chip was further heated at 80 °C for another 2 h to complete thermal bonding.
5
Dissociated Spiral Ganglion Neuron Culture
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Dissociated SGN cultures were conducted in accordance with the procedures from a previous document (Tuft et al., 2013 (link)). The cochlear ducts of P3 rats containing the organ of Corti, spiral ligament, and stria vascularis were sequentially dissected away to collect modiolus tissues harboring the SGNs. The tissues were then applied to enzymatic dissociation by using 0.1% collagenase type IV and 0.25% trypsin in Ca2+/Mg2+-free HBSS (all Thermo Fisher Scientific) at 37°C for 20 min. After enzymatic digestion was inactivated by adding equal volumes of 10% FBS, the tissues were mechanically triturated into cell suspensions through a fire-polished glass Pasteur pipette. The dissociated SG cells were resuspended in neural maintenance medium and seeded at a density of 2.0 × 105 cells/poly-L -lysine-coated (Thermo Fisher Scientific) dish for 4 h.
For neurite outgrowth assay, the attached SGNs were maintained in culture medium supplemented with or without the following reagents consistent with those in SG explant cultures: 20 ng/ml BDNF, 1 μM ANP, 1 μM ANP plus 1 μM A71915, 1 μM 8-pCPT-cGMP, 1 μM ANP plus 1 μM KT5823, 1 μM ANP4–23, or 1 μM ANP plus 1 μM AP811. In each group, five culture dishes containing dissociated cells were replenished with a fresh medium every other day and transferred to a 5% CO2 incubator at 37°C for 5 days before fixation.
For neurite outgrowth assay, the attached SGNs were maintained in culture medium supplemented with or without the following reagents consistent with those in SG explant cultures: 20 ng/ml BDNF, 1 μM ANP, 1 μM ANP plus 1 μM A71915, 1 μM 8-pCPT-cGMP, 1 μM ANP plus 1 μM KT5823, 1 μM ANP4–23, or 1 μM ANP plus 1 μM AP811. In each group, five culture dishes containing dissociated cells were replenished with a fresh medium every other day and transferred to a 5% CO2 incubator at 37°C for 5 days before fixation.
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