Quantifying Membrane Protein Expression

Images were taken with a Nikon A1R + laser scanning confocal microscope system with a 60× NA 1.4 oil immersion objective (Nikon, Vienna, Austria). After aspiration of cell culture medium, cells were incubated with trypan blue (0.4%, Sigma-Aldrich) for 10 min and subsequently washed with KHB. eYFP fluorescence was excited with a 488 nm laser line, trypan blue with a 561 nm laser and monomeric BFP with a 399 nm laser. A 525/50 nm, 595/50 nm or 454/50 nm emission filter was used for eYFP, trypan blue or mTagBFP, respectively. Images were analysed as previously described [38 (link)]. In brief, images, taken on at least three separate days, were analysed with Fiji ImageJ 1.53c. Membrane expression was assessed by drawing the cell membrane and the interior of the call and calculating relative expression at the membrane.