Upon 100x oil objective lens

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.

EV, Rab37^WT, Rab37^Q89L and Rab37^T43N cells were seeded on a 29 mm glass bottom dish. After 24 h, cells were transfected with GFP-SFRP1 for 16–18 h before image analysis. TIRF microscopy system was built on an inverted microscope (Olympus IX81, Tokyo, Japan). The system was equipped with a high sensitivity EMCCD Camera (iXOn3897, Andor technology, New York, NY, USA) and an UPON 100X oil objective lens (NA = 1.49, Olympus) to capture 100–200 nm images below the interface. GFP was excited with 491 nm solid laser and driven by Xcellenace software (Olympus imaging software). To observe the Rab37-mediated SFRP1 trafficking events, the images sequences were recorded in stream model for every 3 s.

TIRF analysis was modified from Tsai's report 24 (link). RAW264.7 macrophage cells were transfected with GFP-CHI3L1 and RFP-Rab37 for 16 h and 1 × 10⁴ cells were re-seeded in 3.5 cm glass bottom dish. TIRF microscopy system (Olympus IX81) equipped with a high sensitivity EMCCD Camera (iXOn3897, Andor technology) and a UPON 100X oil objective lens (NA = 1.49, Olympus) was used to capture 100-200 nm images below the plasma membrane. We defined each green fluorescence spot as a CHI3L1-containing vesicle, and then tracked each vesicle trafficking distance with trackIT software (Olympus).