HEK-293T and COS-7 cell lines were obtained from the American Type Culture Collection and maintained at 37°C in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum and antibiotics [penicillin (100 U/ml) and streptomycin (100 μg/ml)]. Furthermore, d-2-amino-5-phosphonopentanoic acid (Abcam) was added to the medium (final concentrations of 200 or 500 μM for HEK-293T and COS-7 cells, respectively) to avoid excitotoxicity. Transient transfection of HEK-293T cells was achieved by the calcium phosphate method (Clontech), and cell extracts were obtained 48 hours after transfection. COS-7 cells were transfected with Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions, and cells were fixed 24 hours after, for further immunofluorescence analysis.

To prepare dissociated mouse hippocampal neuron cultures, mouse embryos (embryonic day 18) were obtained from pregnant CD1 females, the hippocampi were isolated and maintained in cold Hank’s balanced salt solution (HBSS; Gibco) supplemented with 0.45% glucose (HBSS-glucose). After carefully removing the meninges, the hippocampi were digested mildly with trypsin for 17 min at 37°C and dissociated. The cells were washed three times in HBSS and resuspended in Neurobasal medium supplemented with 2 mM GlutaMAX (Gibco) before filtering in 70-μm mesh filters (BD Falcon). The cells were then plated onto glass coverslips (5 × 104 cells/cm2) coated with poly-l-lysine (0.1 mg/ml; Sigma-Aldrich), and 2 hours after seeding, the plating medium was substituted by complete growth medium, consisting on Neurobasal medium supplemented with 2% B27 (Invitrogen) and 2 mM GlutaMAX, and the coverslips were incubated at 37°C in a humidified 5% CO2 atmosphere. Every 3 to 4 days, half of the conditioned medium was removed and replaced by fresh growth medium. Primary cultures were transfected with 0.8 μg of DNA (Lipofectamine 2000, Invitrogen) on DIV4, DIV7, or DIV11 for further surface expression analysis of GFP-GluN2B constructs and endogenous GluA1. All the experimental procedures were carried out according to European Union guidelines (Directive 2010/63/EU) and following protocols that were approved by the Ethics Committee of the Bellvitge Biomedical Research Institute (IDIBELL).

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