TIRF Microscopy of Rab37 Vesicle Trafficking

The protocol was modified from Kuo’s report [16 (link)]. In brief, 293T (5 × 10⁵) cells were seeded in 3.5 cm glass bottom dish. After 48 h, cells were transfected with RFP-Rab37 or GFP-PD-1 for 18 h. TIRF microscopy system was built on an inverted microscopy Olympus IX81 (Olympus, Tokyo, Japan) equipped with a high sensitivity EMCCD Camera (iXOn3897, Andor technology, New York, United States) and a UPON 100X oil objective lens (NA = 1.49, Olympus) to capture 100–200 nm images below the plasma membrane interface. We defined each green fluorescence spot as a cargo-containing vesicle and trafficking by Rab37 in cells, and then tracked each vesicle trafficking distance with trackIT software (Olympus). The cutoff length of moving vesicle was 3 μm. The vesicle trafficking event was measured as the ratio of moving vesicles to the total vesicles in cells. A total of at least 20 moving vesicles per cell were tracked and 6 cells were scored.