Experiments conformed to the Animals (Scientific Procedures) Act 1986, and were approved by the UK Home Office. Primary cortical neurons were produced from either wild type (C57BL/6J; RRID: IMSR_JAX:000664, purchased from Charles River) or Syt1−/− (B6; 129S-Syt1tm1Sud/J; RRID: IMSR_JAX:002478 The Jackson Laboratory) postnatal day 0 mouse pups of both sexes and cultured in Neurobasal A/B27-based medium (Thermo Fisher Scientific). The cortices were dissected and dissociated by enzymatic digestion in 0.25% trypsin for 10 min at 37 °C and then triturated using a standard p1000 micropipette. Neurons were plated on poly-L-lysine-treated 19-mm glass coverslips (1 mg/mL; Sigma-Aldrich) at a density of ~100,000 cells per coverslip placed in standard 12 well plates. At 5 days in vitro (5 DIV) neurons were transfected with pAAV.hSynap.SF-iGluSnFR.A184V plasmid15 (link) (addgene Plasmid #106174, 450 ng per coverslip) using Neuromag reagent (KC30800; OZ Biosciences). The transfection resulted in sparse expression of the iGluSnFR probe in a small subpopulation of neurons (∼3%), which allowed us to select individual cells for imaging. Experiments were performed between 16 and 21 DIV.
Neurobasal a b27 based medium
Manufactured by Thermo Fisher Scientific
Neurobasal A/B27-based medium is a cell culture medium specifically formulated for the growth and maintenance of primary neuronal cells. It provides a defined, serum-free environment that supports the survival and differentiation of neurons. The medium is composed of the Neurobasal A basal medium and the B27 supplement, which together create an optimized system for culturing neuronal cells.
Automatically generated - may contain errors
3 protocols using neurobasal a b27 based medium
1
Sparse Fluorescent Glutamate Imaging
2
Primary Cortical Neuron Culture from Syt1 Mice
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Experiments conformed to the Animals (Scientific Procedures) Act 1986, and were approved by the ethics committee of the UCL Queen Square Institute of Neurology. Primary cortical neurons were produced from either Syt1+/+ or Syt1−/− mice (B6; 129S-Syt1tm1Sud/J; Jackson Laboratory) and cultured in Neurobasal A/B27-based medium (Thermo Fisher Scientific). Briefly, cortices were dissected from postnatal day 0 pups and dissociated by enzymatic digestion in 0.25% trypsin for 10 min at 37 °C and then triturated using a standard p1000 micropipette. Neurons were plated on poly-l -lysine (1 mg/mL; Sigma-Aldrich)–treated 19-mm glass coverslips at a density of 100,000 to 120,000 cells per coverslip (for imaging and electrophysiological experiments) or on poly-l -lysine (0.1 mg/mL)–treated 35-mm plastic dishes at a density of 500,000 cells per dish for Western blot analysis.
3
Probing Glutamate Dynamics in Mouse Cortical Neurons
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Experiments conformed to the Animals (Scientific Procedures) Act 1986, and were approved by the UK Home Office. Primary cortical neurons were produced from either wild type (C57BL/6J; Charles River) or Syt1 -/-(B6; 129S-Syt1tm1Sud/J; The Jackson Laboratory) postnatal day 0 mouse pups of both sexes and cultured in Neurobasal A/B27-based medium (Thermo Fisher Scientific). The cortices were dissected and dissociated by enzymatic digestion in 0.25% trypsin for 10 min at 37°C and then triturated using a standard p1000 micropipette.
Neurons were plated on poly-L-lysine-treated 19-mm glass coverslips (1 mg/mL; Sigma-Aldrich) at a density of ~100,000 cells per coverslip. At 5 days in vitro (5 DIV) neurons were transfected with pAAV.hSynap.SF-iGluSnFR.A184V plasmid 14 (addgene Plasmid #106174) using Neuromag reagent (KC30800; OZ Biosciences). The transfection resulted in sparse expression of the iGluSnFR probe in a small subpopulation of neurons (∼3%), which allowed us to select individual cells for imaging. Experiments were performed between 16 and 21 DIV. was subtracted from the measurements post hoc. The recorded neuron was held at -70mV and action potentials were evoked using brief voltage steps (5 ms) from -70mV to -10mV, producing stereotypical 'escape' currents.
Neurons were plated on poly-L-lysine-treated 19-mm glass coverslips (1 mg/mL; Sigma-Aldrich) at a density of ~100,000 cells per coverslip. At 5 days in vitro (5 DIV) neurons were transfected with pAAV.hSynap.SF-iGluSnFR.A184V plasmid 14 (addgene Plasmid #106174) using Neuromag reagent (KC30800; OZ Biosciences). The transfection resulted in sparse expression of the iGluSnFR probe in a small subpopulation of neurons (∼3%), which allowed us to select individual cells for imaging. Experiments were performed between 16 and 21 DIV. was subtracted from the measurements post hoc. The recorded neuron was held at -70mV and action potentials were evoked using brief voltage steps (5 ms) from -70mV to -10mV, producing stereotypical 'escape' currents.
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