Ellipse inverted microscope

BEAS-2B cells (~6000 cells/well) were attached in 8-well Ibidi chamber slides (Ibidi, Munich, Germany) for 24 h, then transfected with GCaMP5 plasmid using DNA-In in Opti-MEM I Reduced Serum medium. 24 h after transfection, medium was replaced with fresh BEGM, and imaged in a stage top incubator using a Nikon Ellipse inverted microscope. XY positions of each cell were registered, and medium was replaced with medium containing 0.2 mg/mL menthol. Time-lapse videos were recorded before menthol treatment and 1, 2, 15 and 20 mins after treatment. In some experiments, the TRPM8 receptor antagonist (N(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide, BCTC-10 μM) was used for 20 mins before and during menthol treatment (Sabnis et al 2008 (link)). The change in fluorescence after treatment with/ without inhibitor was recorded using an Andor camera and analyzed using CL Quant software.

BEAS-2B cells (60,000 cells/insert) were allowed to attach for 24 h in transwell inserts followed by transfection with MitoTimer plasmid (Addgene, Cambridge, MA) using DNA-In transfecting reagent according to the manufacturer’s instructions. The cells were incubated with 1 μg of plasmid DNA and 3 μl of DNA In (MTI-GlobalStem, MD) in Opti-MEM medium at 37°C. After 24 h, the transfecting medium was removed from the apical layer and fresh medium was added to the basal layer. The transfected cells were exposed to menthol aerosol made with a solution of 0.8 mg/mL of menthol in 0.8% sodium chloride solution using a VITROCELL® cloud chamber. Untreated control samples remained in the incubator and negative controls were exposed to only 0.8% sodium chloride. 24 h after exposure, cells were fixed on inserts using 4% formaldehyde and insert membranes were transferred to glass slides and mounted using Vectashield with DAPI (Vector Lab, Burlingame, CA). Images were collected using non-saturating exposures on a Nikon Ellipse inverted microscope in the green (excitation/emission 488/518 nm) and red (excitation/emission 543/572 nm) channels.