MDCK Cells Culture and Overexpression

Type II Madin–Darby canine kidney (MDCK) cells were used between passages 3 and 10. These cells were originally cloned by Dr. D. Louvard (European Molecular Biology Laboratory, Heidelberg, Germany) and came to us via Dr. Mostov who obtained them from Dr. K. Matlin (University of Chicago, Chicago, IL). We previously generated MDCK type II cells overexpressing myc‐tagged‐hSec10 (Lipschutz et al. 2000 (link)). MDCK type II cells overexpressing Smoothened‐YFP were generously provided to us by Drs. Ott and Lippincott‐Schwartz (Ott et al. 2012 (link)). Cells were cultured in modified Eagle's minimal essential medium containing Earl's balanced salt solution and glutamine supplemented with 5% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. MDCK cells were seeded at confluence on 24‐mm Transwell filter units coated with collagen (Corning Life Sciences, Lowell, MA). Cell monolayers were used for experiments after 7–14 days of culture with daily changes in medium.