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.
Neuromag reagent
Manufactured by Ozyme
NeuroMag is a magnetic nanoparticle reagent designed for the isolation and purification of neuronal cells and subcellular components. The reagent contains iron oxide nanoparticles that can be used to magnetically separate target cells or organelles from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Calphos mammalian transfection kit, by Takara Bio (4 mentions)
C57bl 6j, by Charles River Laboratories (2 mentions)
Neurobasal a b27 based medium, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Poly l lysine treated, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
60 mm dish, by Corning (1 mentions)
Super magnetic plate, by Ozyme (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
12 protocols using neuromag reagent
1
Sparse Fluorescent Glutamate Imaging
2
Lentiviral Transduction of Neuronal Cultures
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The production of vesicular stomatitis virus-pseudotyped second- and third-generation lentiviruses was performed by cotransfection of the pFUGW/L309/PCCL-based expression vectors and 2 (pCMVdelta and pCMV-VSV-G) or 3 helper plasmids (pCMV-VSV-G, pMDLg/pRRE, pRSV-Rev) in human embryonic kidney 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific) following the instructions of the manufacturer. Primary neurons were transduced at 7 d in vitro (DIV) using 10 multiplicity of infection either with viruses expressing the Myc-tagged Syt1WT and Syt1F349A (Figs. 1 and 3 ) or the mCherry-tagged Syt1WT and Syt1F349A. Transduction efficiency (80 to 95% range) was verified using immunofluorescence analysis (SI Appendix, Figs. 2–5 ). For experiments in Fig. 1 , neuronal cultures were also transduced with the sypHy-expressing lentivirus. For experiments in Fig. 2 , cortical neurons were transfected at 5 DIV with the pAAV.hSynap.SF-iGluSnFR.A184V plasmid using Neuromag reagent (KC30800; OZ Biosciences). This allowed expression of the iGluSnFR probe only in a small (∼3 to 5%) subpopulation of neurons, which was essential for imaging of vesicular release in individual synaptic boutons.
3
CRISPR Knockdown of GluN2A/B in Neurons
4
Receptor Expression and Live Imaging in Neurons
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HEK293T cells were transfected with α5β3γ2 receptors, together with GFP or Shisa7/GFP, using CalPhos Mammalian Transfection Kit (Takara). Electrophysiological recordings or immunostaining were performed 24–48 h after transfection. Hippocampal neurons at DIV13–15 were transfected with SEP-α5 using Lipofectamine 3000 (Thermo Fisher Scientific) and live imaging was performed 24 h after transfection. Hippocampal neurons at DIV16 were transfected with Shisa7 phosphomimic (S405D), phosphodeficient (S306A, S405A, S430A) or ΔC-tail mutants for overexpression and rescue experiments using NeuroMag reagent (Oz Biosciences). Electrophysiological recordings or immunostaining were performed 36–72 h after transfection. All transfection kits were used according to the manufacturer’s instructions.
5
Efficient Magnetic Transfection of iPSC Cells
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All transient transfection was carried out using magnetofection protocol by Oz Biosciences using their NeuroMag reagent. Briefly, differentiated iPSC cells were grown in a 12‐well plate and the media was changed on the day of transfection with 1 ml of antibiotic‐free media. The cells were transfected first on day 25 of differentiation. Around 3 µg of total plasmid DNA was transfected per well following equal molar mass for all three vectors (0.16 pmols each). The DNA was then diluted in 150 µl of Opti‐MEM media and spun down. The diluted DNA was mixed gently with 9 µl NeuroMag reagent in a different tube and allowed to incubate for 30 min before adding to the cells. After adding the transfection mix to the cells, the plate was put on a magnetic bar overnight in the 37°C incubator, and the next day, the magnet was removed underneath the plate. This process ensured a very high transfection rate without any visible cell mortality. Transgene expression was visualized by expression of sfGFP associated with the SunTag system. To increase transfection efficiency, cells were transfected again the same way after 2 days of the first transfection and harvested 6 days after first transfection.
6
Isolation and Transfection of Mouse Embryonic Neural Stem Cells
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Mouse embryonic neural stem cells were isolated from E14 cortices using NeuroCult Proliferation Kit Mouse (STEMCELL Technologies) and maintained as recommended. Primary neurons were prepared from E15.5 mouse cortices and cultured essentially as in ref. 58 (link), except no astroglial feeders were used for cultures maintained ≤7 days in vitro (DIV). Neurons were transfected using NeuroMag reagent (Oz Biosciences) as recommended. Briefly, 2.4 × 106 cortical neurons were plated per 60-mm dish (Corning) pretreated with poly-D-lysine (Sigma). At DIV2–DIV5, 0.1 μg of TTP expression plasmid (pBS-CMV-TTP) or corresponding vector control or 4 μg of a dTomato reporter plasmid (dTom-3′HuR-pA2mut or dTom-3′HuR-pA2mut/ΔARE) was incubated with 6 μl NeuroMag beads in 150 μl Opti-MEM I for 20 min and the mixture was added dropwise to the dish containing neurons. The cultures were then incubated on top of Super Magnetic Plate (Oz Biosciences) for 40 min and incubated for another 40–48 h at 37 °C and 5% CO2 before subsequent analyses.
7
Receptor Expression and Live Imaging in Neurons
8
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.
9
Overexpression of VAPB Proteins
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Cells were transfected to express either GFP-wt-VAPB or GFP-P56S-VAPB. The detailed description of generation of these plasmids is given elsewhere31 (link). The empty pEGFP-N1 vector was used as a transfection control. All cell lines were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s recommendations. After 4 h incubation at 37 °C and 5% CO2 the transfection reagent containing medium was replaced by fresh medium and analysis was performed 48 h later. IPS-derived MNs were transfected using NeuroMag reagent (OZ Biosciences) according to the manufacturer’s protocol; the analysis was performed after 48 h.
10
CRISPR Knockdown of GluN2A/B in Neurons
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HEK293T cells were transfected with GluN2A or GluN2B, together with sgRNA using CalPhos Mammalian Transfection Kit (Takara). Western blot was performed 48 h after transfection. Hippocampal neurons at DIV3–4 were transfected with GluN2A sgRNA or GluN2B sgRNA using NeuroMag reagent (Oz Biosciences), and were recorded at DIV16–17. Hippocampal neurons at DIV11 were co-transfected with pCAG-IRES-GFP and GluN2A or GluN2B using NeuroMag reagent. Electrophysiological recordings or immunostaining were performed 72 h after transfection. All transfection kits were used according to the manufacturer’s instructions.
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