Poly d lysine coated 35 mm glass bottom imaging dishes

Mouse neuroblastoma 2a (neuro-2a) cells were cultured in the following medium: MEM with 5% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells were plated by adding 90,000 or 50,000 cells (microscopy and electrophysiology, respectively) to poly-D-lysine-coated 35-mm glass-bottom imaging dishes (MatTek Corporation) and cultured in a humidified incubator (37°C, 95% air, 5% CO₂). Cells were transfected with 500 ng of each nAChR subunit cDNA plasmid (α4-mCherry, α4-GFP, and β2wt or α4-mCherry, α6-GFP, and β2wt depending on intended subtype target and FRET pairing). Following plating procedures, plasmid DNA was mixed with 250 μl of Opti-MEM and Lipofectamine-3000 was separately added to the Opti-MEM. After 5 min at 24°C, the two solutions were combined and incubated at 24°C for 25 min. Plated neuro-2a cells then received the mixed solution and were incubated for 24 h. The next day, Opti-MEM was removed, and the cells received growth medium. 500 nm filter-sterilized farnesene (or sham treatment) was added after replacing the Opti-MEM with standard culture medium. Twenty-four hours after drug/sham addition, neuro-2a cells were imaged on a confocal microscope (discussed above) or examined using electrophysiological methods (discussed below).

Neuro-2a cells were cultured using standard techniques (Srinivasan et al., 2011 (link)). For imaging, cells were plated by adding 90,000 cells to poly-d-lysine-coated 35 mm glass-bottom imaging dishes (MatTek) and cultured in a humidified incubator (37°C, 95% air, 5% CO₂₎. Cells were transfected as described previously (Henderson et al., 2014 (link)). Similar to previous assays (Henderson et al., 2016 (link)), drug treatments (control or 500 nm menthol stereoisomers) were applied for 24 h. Culture medium containing menthol was removed 1 h before the total internal reflection fluorescence microscopy (TIRFM) assays and replaced with extracellular solution (ECS), identical to methods previously described (Henderson et al., 2016 (link), 2017 (link)).