Wildtype mice were mated and at e15 the females were euthanized and the embryos were removed. The females were not further used. The embryos were decapitated and cortex tissue was dissected and used for primary cultures. Culture set-up is previously described in Perland et al. (2016) (link).Briefly, the cortex samples were pooled, washes in PBS-Glucose and dissociated using papain (Thermo Fisher Scientific) and DNAse (Thermo Fisher Scientific) for 30 min. Thereafter, mechanical dissociation was performed by pipetting before filtering the cell solution through a cell strainer (Thermo Fisher Scientific). The cells were diluted in plating media consisting of DMEM:F12 (Gibco, Invitrogen) supplemented with 2 mM GlutaMax, 1 mM Na-Pyruvate, 10% FBS and 1% Pen strep, all supplied from Invitrogen, and plated on Poly-L -Lysine (Sigma) coated coverslips (12 mm, #1.5) (Menzel-Gläser, Thermo Fischer Scientific) in 24-well plates (Nonclone delta, Thermo Fischer Scientific) or on Poly-L -Lysine coated 6-well plates (Nunclone delta, Thermo Fischer Scientific). The cell was incubated at 37°C, 5% CO2 for 3 h before media change to NeurobasalA media (Gibco, Invitrogen) supplemented with 2 mM Glutamax, 1 mM Na-Pyruvate, 1 % Pen-strep and 1x B27 Supplement. 75% of the media was changed every third day and the cells allowed to grow for 9 days.
Poly l lysine (pll)
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Poly-L-lysine is a synthetic amino acid polymer used in various laboratory applications. It serves as a cell adhesion promoter, facilitating the attachment of cells to surfaces. This product is commonly utilized in cell culture, histology, and related research fields.
Automatically generated - may contain errors
Lab products found in correlation
Poly l lysine (pll), by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Laminin, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Neurobasal medium, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
B27 supplement, by Thermo Fisher Scientific (4 mentions)
N2 supplement, by Thermo Fisher Scientific (3 mentions)
Neurobasal, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Glutaraldehyde, by Merck Group (3 mentions)
L glutamine, by Merck Group (2 mentions)
Poly l lysine solution, by Merck Group (2 mentions)
Gentamicin, by Merck Group (2 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
87 protocols using poly l lysine (pll)
1
Primary Mouse Cortical Neuron Culture
2
Isolation and Culture of Primary Cortical Neurons
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Experiments involving animals were approved by the Institutional Animal Care and Use Committee of Capital Medical University of Science and Technology (Beijing, China; approval no. SCXK-2011-004) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication no. 80-23). Primary cortical neurons cultured as previous described.32 (link) Briefly, surgeries were performed under sodium pentobarbital anesthesia. Primary cortical neurons were prepared from Sprague-Dawley rats or C57BL mouse E14.5–E15.5 embryos and cultured in 3.5cm dishes (1.4 × 106 (link) cells/dish) on cover slips coated with 100 μg/ml poly-L -lysine in neurobasal medium (21103–049; Invitrogen) supplemented with basic fibroblast growth factor (10 ng/ml), nerve growth factor (10 ng/ml), L -glutamine (0.5 mM) and B27 supplement. After 7 days, primary neurons were infected with LV gene transfer vectors.
3
Isolation of Primary Rat Microglia
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The preparation of mixed glial cell cultures and the isolation of microglia were carried out according to Saura et al[38 (link)]. The brains of neonatal (P1–P3) rats were isolated, then the meninges and choroid plexus were removed. The rat cortices were digested by 0.05% trypsin–EDTA for 15 min at 37°C. After centrifuging at 1200 rpm for 2 min, the cells were plated in 75-cm2 flasks that had been coated with poly-L-lysine (Invitrogen). After 48 h, the nonadherent cells were removed by thorough washing of the surface with culture medium. Mixed glial cells were cultured in DMEM supplemented with 10% FBS, 2 mmol/L L-glutamine (Sigma–Aldrich), and 1% penicillin/streptomycin at 37°C in 5% CO2 in air and 95% humidity for 2 wk. The culture medium was changed every 3 d. To harvest pure microglia, microglia on the confluent mixed glial cell layer were isolated by shaking the flasks for 2 h at 180 rpm. The medium containing the layer of detached microglia was collected and immediately centrifuged for 2 min at 1200 rpm. The supernatant was removed, and the obtained pure microglia were resuspended in fresh culture medium and seeded into subcultures. Microglial harvesting was repeated for a maximum of three times at intervals of 3 d. The purity of our primary microglia culture was assessed by immunocytochemical staining for ionized calcium-binding adapter molecule (Iba1).
4
Nanoparticle-based Cell Proliferation Assay
5
Cortical and Hippocampal Neuron Culture
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Cortical and hippocampal neuronal cultures were carried out following published protocols99 (link). Briefly, cortical or hippocampal tissues were dissected from + /+, fl/+, fl/fl E18 mouse embryos and extensively rinsed in HBSS with 1% P/S, followed by dissociation in 0.25% trypsin with 1 mg ml−1 DNase I. Neurons were isolated and plated with plating media (Neurobasal media with 10% FBS, 1xB27,1xGlutaMAX) onto glass coverslips at appropriate density. Both the coverglasses and plates were pre-coated with 0.1% poly-L-lysine (Invitrogen). Plating medium was replaced with maintenance medium (Neurobasal media, 1xB27, 1xGlutaMAX) the following day. Only 2/3 of the media was replaced every other day until the conclusion of the experiments.
6
Transfection of Murine Embryonic Fibroblasts
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MEFs were cultured in antibiotic-free culture medium at a density of 80 000 cells/ml on glass slides (diameter: 40 mm) coated with poly-l -lysine (Invitrogen). 18–24 h later, MEFs were transfected by using either TransFectin Lipid Reagent (Biorad, Hercules, CA, USA) or FuGENE HD Transfection Reagent (Promega, Madison, WI, USA) according to the manufacturer's protocols. Medium was replaced with regular culture medium 4–6 h after transfection, and MEFs were used for experiments 24 h after transfection. Generation of constructs has been described previously: mtYFP and mtRFP.66 (link)
7
Evaluating Radiosensitivity of Glioma Cells
8
Isolation of Rat Cortical Microglia
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Cerebral cortices of newborn Sprague Dawley rats (Institute of Neuroimmunology, Bratislava, Slovakia) (1 day old) were dissected by cervical dislocation, stripped of their meninges, and mechanically dissociated by repeated pipetting followed by passing through a nylon mesh. Cells were plated in 96-well plates and 75 cm2 flasks pre-coated with poly-L-lysine (10 mg/ml) and cultivated in DMEM containing 10% FCS and 2 mM L-glutamine (all from Life Technologies Invitrogen, Carlsbad, California, United States) at 37°C, 5% CO2 in a water-saturated atmosphere. The medium was changed twice a week. Mixed glial cultures reached confluence after 8 to 10 days in vitro. Confluent mixed glial cultures were subjected to mild trypsinization (0.05 to 0.12%) in the presence of 0.2 to 0.5 mM Ethylenediaminetetraacetic acid (EDTA). This resulted in the detachment of an intact layer of cells containing astrocytes, leaving undisturbed a population of firmly attached cells identified as more than 98% microglia [40 (link)]. The cells were maintained in astrocyte-conditioned medium and were used for experiments after 24 hours in culture. The purity of microglial cell cultures isolated by this procedure was 95% (CD11b/CD18 staining).
9
Isolation of DRG Neurons for Experiments
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DRG neurons were isolated using protocols described by our previous experiments [5 (link)]. Briefly, The L4–L6 DRGs were collected in cold DH10 (90% DMEM/F-12, 10% FBS, and 1% penicillin-streptomycin-glutamine; Invitrogen) and treated with enzyme solution (5 mg/ml dispase, 1 mg/ml collagenase type I in Hanks' Balanced Salt Solution without Ca2+ and Mg2+, Invitrogen) at 37°C. Following trituration and centrifugation, cells were resuspended in DH10 and nerve growth factor (50 ng/ml, Upstate), plated on poly-L-lysine (0.5 mg/ml, Stoughton, MA, USA) and laminin (10 μg/ml, Invitrogen) coated glass coverslips, cultured in an incubator (95% O2 and 5% CO2) at 37°C and used within 48 hours.
10
Culturing Cerebellar Granule Neurons
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Cerebellar granule neurons (CGNs) were dissociated from 7- to 9-day-old Sprague–Dawley rat cerebellum and purified as previously described [50 (link),51 (link)]. Briefly, the cerebella were triturated to dissociate the neurons and plated onto coverslips treated with poly-L-lysine (1 µg/mL) (Invitrogen Life Technologies, Carlsbad, CA, USA) at a density of 2.5 × 105 cells/cm2. Cells were then incubated at 37 °C in a 5% CO2 in DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCS (fetal calf serum), 25 mM KCl, 39 mM glucose, 5 mM glutamine, and 1% penicillin and streptomycin. After 4 days, the culture medium was exchanged. The experiments with CGNs were performed between 7 and 14 days in culture. The experimental procedures were approved by the Institutional Bioethics Committee (CIECUAL) of the University of Talca (Code: CIECUAL-UTALCA 22-01).
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