E14.5 or E15.5 mouse cortices were digested in 0.25% trypsin (Thermo Fisher Scientific) with DNase (1:1000, Sigma, Merck, Darmstadt, Germany) for 20 min at 37 °C, followed by dissociation in DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum, as described previously.19 (link) The cells were washed and resuspended in 100 μl of Mouse Neuron Nucleofector Solution (Lonza, Basel, Switzerland) with 5 μl of 25 μM NCP, miR484P, NCI or miR484I. Immediately after electroporation, the cells were mixed with 500 μl of DMEM/F-12 containing 10% fetal bovine serum, and the cell suspension was transferred onto poly-l -lysine-coated dishes. The medium was exchanged with DMEM/F-12 (Thermo Fisher Scientific) containing 2% B27 supplement (Thermo Fisher Scientific) 12 h later.
Mouse neuron nucleofector solution
Manufactured by Lonza
Sourced in Switzerland
The Mouse Neuron Nucleofector Solution is a specialized laboratory reagent designed for the transfection of mouse neurons. It is used to efficiently introduce nucleic acids, such as plasmids or small interfering RNAs, into mouse neuronal cells.
Automatically generated - may contain errors
3 protocols using mouse neuron nucleofector solution
1
Electroporation of Mouse Cortical Neurons
2
Primary Mouse Cortical Neuron Culture
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E15.5 mouse cortices were digested in 0.25% trypsin (Thermo Fisher Scientific) supplemented with DNase (1:1000, Sigma) for 20 min at 37 °C. Cells were dissociated in DMEM containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 120 mg/ml penicillin, and 200 mg/ml streptomycin sulfate. Then, cells were washed and resuspended in 100 μl of Mouse Neuron Nucleofector Solution (Lonza) containing 3 μg of plasmid DNA or shRNA vector. After electroporation using a Nucleofector kit (Lonza), the cells were immediately mixed with 500 μl of DMEM/F-12 containing 10% FBS, and the cells were plated onto poly-L-lysine-coated dishes and cultured at 37 °C in the presence of 5% CO2. The medium was exchanged with DMEM/F-12 (Thermo Fisher Scientific) containing 2% B27 supplement (Thermo Fisher Scientific) 12 h later.
3
Dissociation and Knockdown of Mouse Cortical Neurons
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E16 mouse cortices were digested in 0.25% trypsin (Thermo Fisher Scientific) supplemented with DNase (1:1000, Sigma) for 15 min at 37 °C. Cells were dissociated in DMEM/F12 (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific). Cells were then washed and resuspended in 100 μL of Mouse Neuron Nucleofector Solution (Lonza) containing 3 μg of Nrp1 shRNA or control shRNA (points-mutated Nrp1 shRNA) vector. The shRNA sequences were as follows: Nrp1, 5′-AGAGAAGCCAACCATTATA-3′; control, 5′-AGAGAAGACAGCCCTTATA-3′37 (link). After electroporation using a Nucleofector kit (Lonza), the cells were immediately mixed with 500 μL of DMEM/F-12 containing 10% FBS, and the cells were plated onto poly-l -lysine-coated dishes and cultured at 37 °C in the presence of 5% CO2. The medium was exchanged with DMEM/F-12 (Thermo Fisher Scientific) containing 2% B27 supplement (Thermo Fisher Scientific) 12 h later.
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