Lysotracker

Dilute exosomes-containing solution at 1:20 dilution with PBS. Add α-syn antibody (Abcam, ab138501) to solution to get 1:200 dilution, and incubate at room temperature on a PLL-coated coverslip for 90 min. Wash with PBS three times. Add Alexa Fluor 750 labeled goat anti-rabbit IgG at 1:1000 dilution, and incubate at room temperature on a PLL-coated coverslip for 90 min. Stain with 0.1 M DiD (a flurescent lipophilic dye) for 5 min. Wash with PBS three times. Observations were made with super-resolution microscopy.
We placed 8 mm-diameter coverslips inside a 12-well plate, and seeded human microglia (HM) (ScienCell, Human Microglia, Catalog # 1900) cells into the wells. On the following day, we made a freshly prepared medium containing with 2 μM SiR-tubulin (Cytoskeleton, Catalog # CY-SC002), removed the coverslips and placed them inside the freshly prepared medium. Subsequently they were incubated in 37 °C for 30 min. For the last 3 min, we added 1 μM lysotracker (Beyotime, C1046) inside the wells. After staining, the cells were washed two times with fresh Dulbecco's Modified Eagle's Medium (DMEM) and then observed by imaging. After searching for some target cells in the region of interest, we added 100 μl PKH67-labeled exosomes inside the light-sheet chamber (the buffer volume was around 4 ml), and then observed using imaging for 60 min (the imaging speed was 1 min per frame).

Gene-edited cells were seeded in 24 well plates at a density of 5 × 10⁵ cells per well for 24 h. Lysotracker (Beyotime, C1046) was diluted in DMEM medium at a ratio of 1:1000. After staining for 10 min, the Hoechst nuclear stain (Thermo) was added to the medium at a ratio of 1:500. After incubating for 10 min at 37 °C, the cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. The fluorescence intensity was observed using a confocal microscope, and representative cells were selected and photographed.

The lysosomal acidification was estimated using LysoTracker, following the manufacturer’s instructions (C1046, Beyotime, China). The fluorescence intensity was observed under confocal laser scanning microscope (ZEISS LSM 800) and representative cells were selected and photographed.

After 48 h of transfection with IFITM3 siRNA, cells were treated with or without 100-nM rapamycin (RAMP, Selleck, USA) for 4 h then analyzed after incubation with 50-nM LysoTracker (Beyotime, China), 50-nM MitoTracker (Beyotime) and 1-μM ER-Tracker (Beyotime) for 30 min. The nuclei were stained with Hoechst 33342 (Sigma-Aldrich, Germany).

The cells of each group were inoculated into 24-well plates with 1 mL of Mito-Tracker (C1048, Beyotime, China) working solution and incubated for 30 min at 37 °C in a 5% CO₂ incubator for mitochondrial staining; 1 mL of Lyso-Tracker (C1046, Beyotime, China) working solution was added to the cells before being incubated for 30 min at 37 °C in a 5% CO₂ incubator. The cells were washed, observed using fluorescent microscopes, and photographed. The fluorescence intensity was analyzed by ImageJ software.

The washing buffer (5 $\times$ 10^− 3 м MgCl₂ and 4.5 g L^− 1 glucose in Dulbecco’s PBS) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and the binding buffer was composed with yeast tRNA (0.1 mg mL^− 1, Sigma-Aldrich) and bovine serum albumin (BSA; 1 mg mL^− 1 (ThermoFisher Scientific, Waltham, MA, USA) in washing buffer, which was used to reduce background binding. Super GelRed was purchased from US Everbright® Inc (Sayreville, NJ, USA). Beijing Labor Technology Co, Ltd. provided the Cell Counting Kit-8 (CCK-8) (Beijing, China; cat. no. 21,162,196). Protein markers were purchased from Sangon Biotech Co. Ltd. (Shanghai, China). The anti-PTK7 rabbit monoclonal antibody was purchased from Abcam (Cambridge, MA, USA). The Alexa Fluor 488-labeled goat anti-mouse secondary antibody, Hoechst 33,342, lysotracker, radioimmunoprecipitation assay (RIPA) lysis buffer, phosphatase inhibitors cocktail, protease inhibitors, and annexin V-FITC Apoptosis Detection Kit (no. 011821210207) were all purchased from Beyotime Biotech Inc (Jiangsu, China). All chemicals for synthesis and purification were purchased from Energy Chemical (Shanghai, China). All tubes and plates for cell culture were purchased from NEST Biotechnology (Jiangsu, China). Unless otherwise stated, all other bioreagents were purchased from Sigma.