Opti mem 1x

Chemical transfections of cultured neurons were performed at 37°C with 5% CO₂ in NB plus supplements media without P/S. First, 7 μl of Lipofectamine 2000 (LFA2K) (Thermo Fisher Scientific) was added to 100 μl Opti-MEM 1X (GIBCO), and plasmid DNA was added to 50 μl of Opti-MEM (per dish). LFA2K and plasmid DNA were mixed into 1 tube and incubated for 20–30 min at 37°C. For each dish, conditioned media was replaced with antibiotic-free NB and the DNA complexes were added and incubated for 4 hours, when the media was replaced with half conditioned media and half fresh NB plus supplements. Dishes were incubated for 2–3 days, depending on the experiment, to allow for adequate expression of plasmid-encoded fluorescent proteins or shRNAs. For rapid SIM studies, we used 3.5 μg of DNA pDsRedExpress2-N1 (Clontech). For confocal studies of the PSD and spinule dynamics, we used 3.5 μg of p-mRuby-N1 plasmid, a gift from Michael Davidson (Addgene #54581) + 3.5 μg of pCAG_PSD95.FingR-eGFP-CCR5TC, a gift from Don Arnold (Addgene #46295). For Ca²⁺ transients experiments, we used 3.5 μg p-mRuby-N1 + 3.5 μg pGP-CMV-GCaMP6s, a gift from Douglas Kim, GENIE Project (Addgene #40753). Four μg of p-mRuby-N1 alone was used in the FM dye labeling experiments, and pGFP, a gift from Stephen Mayfield (Addgene #64904), was added at 2 μg per neuronal coverslip for fixed imaging experiments.