SUPPORTING MATERIAL STED Microscopy with Optimized Labeling Density Reveals 9-fold Arrangement of a Centriole Protein

IMCD3 cells were grown in DME/F12 (Invitrogen) supplemented with 10% FBS at 37°C and 5% CO2. Mouse tracheal epithelial cell (MTEC) cultures were established as previously described (1, 2). Mice were sacrificed at 2–4 months of age, and trachea were excised, opened longitudinally to expose the lumen, and placed in 1.5 mg/ml pronase E in F12-Kaighn’s media (Invitrogen) at 4°C overnight. Tracheal epithelial cells were dislodged by gentle agitation and collected in F12-Kaighn’s with 10% FBS. After centrifugation, cells were treated with 0.5 mg/ml DNase I for 5 min on ice and centrifuged at 4°C for 10 min at 400 g. Cells were resuspended in DME/F12 with 10% FBS and plated in a tissue culture dish for 3 h at 37°C with 5% CO2 to adhere contaminating fibroblasts. Non-adhered cells were collected, concentrated by centrifugation, resuspended in an appropriate volume of MTEC-Plus medium (as described in (1)), and seeded onto Transwell-clear permeable filter supports (Corning). The air-liquid interface was established 2 days after cells reached confluence by feeding MTECs serum-free medium (1) only in the lower chamber. Cells were cultured at 37°C with 5% CO2, and media were replaced every 2 days. All chemicals were obtained from Sigma-Aldrich unless otherwise indicated. All MTEC media were supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml Fungizone (all obtained from Invitrogen).