Glass bottom fluorodishes

Neurons and support cells from dissociated ganglia were plated on laminin-coated (Life Technologies, Frederick, MD) glass-bottom fluorodishes (World Precision Instruments, Saratoga, FL) and cultured in media consisting of 1:1 F12:DMEM media (Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml guinea pig transferrin (BP25445, FisherScientific), 100 ng/ml nerve growth factor 2.5S (NG009, Sigma-Aldrich, St. Louis, MO), and 20 μM camptothecin (Sigma-Aldrich, St. Louis, MO) for 48 h. Cells were loaded with a nitric oxide-detecting fluorophore (FL2A, Strem Chemicals) for 30 min followed by treatment with TLR7 agonist R837 (10 μM, Invitrogen) for an additional 30 min. Some cells were pre-treated with TLR7 antagonist IRS661 (100 μM, Integrated DNA Technologies), TLR7 antagonist control oligomer (100 μM, Integrated DNA Technologies), or the nitric oxide synthase antagonist L-NAME (100 μM, Sigma). Cells were imaged on a Nikon spinning-disk confocal microscope (× 20, 1.3 NA), and cellular fluorescence was quantified within individual cell bodies using ImageJ. Experimental replicates represent the average of 10–20 individual cells per experimental condition.

Cells were plated, at a density of 3000 cells/cm², on glass-bottom fluorodishes (World Precision Instruments) coated with collagen. Cells were transfected 48 h before imaging using Leica SP5 confocal microscope with a 63X objective. Nuclei were labeled with Hoechst staining (blue). A TMEM33-EGFP construct was stably expressed in Hela cells. Primary cilia were visualized with acetylated tubulin antibody (Sigma T7451; 1/200) and a secondary donkey anti-mouse antibody (Alexa 647 Invitrogen A-31571; 1/500). ER was marked with ER Tracker blue (invitrogen E12353) and lysosomes by Lysotracker red (invitrogen L7528).