Menthol Effects on Neuro-2a Cells

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)).