Electroporation and transfection

For imaging of DIV (day in vitro) 1 to 3 neurons, electroporation was performed, whereas for imaging of neurons >DIV3, Lipofectamine transfection was used. Electroporation of hippocampal neurons was performed directly after dissociation. The dissociated neuron suspension was spun down at 200g for 5 min, the supernatant was removed, and neurons were resuspended carefully in electroporation buffer [12.5 mM NaCl, 123 mM KCl, 20 mM KOH, 10 mM EGTA, 4.5 mM MgCl₂, and 20 mM Pipes (pH 7.2)] mixed with 20% fetal calf serum (FCS). The electroporation buffer and FCS aliquots were stored at −20°C and mixed after being thawed separately. Vigorous resuspension using a pipette can damage the neurons, and small clumps of neurons that did not resuspend after four to five times mixing with the pipette were removed from the solution whenever possible. The resuspended neurons were mixed with 1 to 3 μg of plasmid DNA and placed in electroporation cuvette (density of 200,000 to 1 million neurons per cuvette) (Bio-Rad, GenePulser; 0.2-cm gap) and electroporated in a Nucleofector 2b device (Lonza) on program O-003/rat hippocampal neurons. After electroporation, neurons were diluted in full medium and divided over multiple wells at ~50,000 neurons per 18-mm coverslip, which were coated as described above. For DIV1 neuron imaging, the following amounts of DNA were used: K353/K560 (1.5 to 2 μg), TRIM46 (0.8 μg), K560Rigor (0.3 μg), iLID-CAAX (1 μg), PA-tubulin (2 μg), mRFP-fill (0.3 μg), and PA-Rac1 (1 μg). For Lipofectamine transfections, 50,000 neurons per well of a 12-well plate were directly plated in full medium onto 18-mm coverslips coated as described above and transfected at the indicated times using Lipofectamine 2000 (Invitrogen). For 1 well of a 12-well plate, 1.8 μg of DNA was mixed with 3.3 μl of Lipofectamine in 200 μl of NB and incubated for 30 min at room temperature. Meanwhile, the conditioned medium, the medium in which neurons were cultured, was transferred to a new plate, and transfection medium (NB medium supplemented with 0.5 mM glutamine) was added. The DNA/Lipofectamine mix was added to the neurons in transfection medium and incubated for 45 min at 37°C and 5% CO₂. Neurons were rinsed by dipping coverslips in prewarmed NB medium and placed back into conditioned full medium for 1 to 2 days before imaging.

U2OS cells were purchased from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium containing 10% FCS and penicillin/streptomycin (50 μg/ml) at 37°C and 5% CO₂. Cells were confirmed to be free of mycoplasma. U2OS cells were plated on 25-mm-diameter coverslips 2 days before transfection. For transfection of one well from a six-well plate with a 25-mm coverslip, 6 μl of FuGENE 6 transfection reagent (Promega) was added to 100 μl of Leibovitz’s L-15 medium followed by addition of 0.3 to 2 μg of DNA. The mix was incubated for 5 min at room temperature and then homogeneously added to the medium in a well.

Developmental stages of neurons were categorized as stage 2, stage 3, and stage 4 by morphology (4). DIVs were counted after seeding the dissociated neurons: DIV1 to DIV2 (26 to 48 hours), DIV2 to DIV3 (48 to 72 hours), and DIV7 to DIV8 (168 to 192 hours) neurons were used for stage 2, stage 3, and stage 4 of neuron development, respectively (4, 12).