HEK293 cell transfections

Plasmid DNAs (Supplementary Data ¹⁵ and Supplementary Data ¹⁷) were purified in ddH₂O for transfections. pGP-CMV-GCaMP6f was a gift from Douglas Kim & GENIE Project (Addgene plasmid #40755; [http://n2t.net/addgene:40755]; RRID: Addgene_40755). Wild-type HEK293 cells (ATCC CRL-1573) were cultured on 24-well plates in DMEM media (4.5 g L⁻¹ d-glucose, l-glutamine; ThermoFisher #11965-092) supplemented with 10% FBS and 1% Penicillin/Streptomycin in a 5% CO₂ atmosphere at 37 °C. Cells were seeded into 24-well plates (ThermoFisher #{"type":"entrez-nucleotide","attrs":{"text":"FB012929","term_id":"159416269"}}FB012929) at 100,000 cells well⁻¹. Twenty-four hours after seeding, cell media was aspirated and replaced with 200 μL of DMEM media. Lipofectamine 3000 (ThermoFisher Scientific) reagents were prepared according to the manufacturer’s instructions by mixing 1.50 μL of Lipofectamine 3000 reagent diluted in 25.0 μL of OPTI-MEM with 1 μg of plasmid DNA diluted in 25.0 μL OPTI-MEM and 2.00 μL of P3000 reagent, allowing for formation of DNA–lipid complexes for ~10 min after combination of the two mixtures. Reagent volumes were increased for the number of wells to be transfected. Fifty microliters of complexed Lipofectamine reagents were pipetted into each well, cells were incubated for 30 min, and the volume in each well was raised to 750 μL with DMEM media. Approximately 6 h after transfection, media was replaced with 1.00 mL of DMEM media. Cells were incubated for 48 h to allow for protein expression.