Cells were plated in Lab-Tek II chambered coverglass and transfected as described above. For cells transfected with HaloTag-Sec23A, 0.2 μM of JF635 HaloTag ligand was added to the cell culture medium 1 hr before imaging. After incubation at 37°C for 30 min, the cells were rinsed with the normal cell culture medium for 5 min × 6 times. Prior to imaging, 25 mM HEPES was added to the cell culture medium to maintain the pH in the ambient environment.
Live-cell fluorescence microscopy was performed on an Olympus IX73 inverted epifluorescence microscope with a water-immersion objective (Olympus, UPLSAPO60XW, NA 1.2) and a mercury lamp, or a Nikon Eclipse Ti-E inverted fluorescence microscope with an oil-immersion objective (Nikon CFI Plan Apochromat λ 100×, NA 1.45) with 488-nm, 560-nm, and 647-nm lasers modulated by an acousto-optic tunable fiber (AOTF, Gooch & Housego, 97–03151-01). Cells were imaged at 2–20 frames per second (fps) at room temperature to moderately slow down the motion of the fast-moving t-ERGIC. Concurrent multi-color imaging was achieved by modulating the AOTF to allow frame-synchronized alternating excitation at 488, 560, and 647 nm (Yan et al., 2020 (link)) with a multi-bandpass filter cube (Semrock Di01-R405/488/561/635 and Chroma ZET405/488/561/640m).
Live-cell fluorescence microscopy was performed on an Olympus IX73 inverted epifluorescence microscope with a water-immersion objective (Olympus, UPLSAPO60XW, NA 1.2) and a mercury lamp, or a Nikon Eclipse Ti-E inverted fluorescence microscope with an oil-immersion objective (Nikon CFI Plan Apochromat λ 100×, NA 1.45) with 488-nm, 560-nm, and 647-nm lasers modulated by an acousto-optic tunable fiber (AOTF, Gooch & Housego, 97–03151-01). Cells were imaged at 2–20 frames per second (fps) at room temperature to moderately slow down the motion of the fast-moving t-ERGIC. Concurrent multi-color imaging was achieved by modulating the AOTF to allow frame-synchronized alternating excitation at 488, 560, and 647 nm (Yan et al., 2020 (link)) with a multi-bandpass filter cube (Semrock Di01-R405/488/561/635 and Chroma ZET405/488/561/640m).