Fv1000 ix81 microscope

Cultured neurons were co-infected with EGFP-CLSTN1 and ICAM5-mCherry lentivirus on DIV 3 and time-lapse imaged using Olympus FV1000-IX81 microscope (FluoView1000-IX81, Olympus, Tokyo, Japan) 8 days after co-infection with the following parameters: 100× (1.4 NA), zoom 4, and 512 × 512 frame size; 12 bit depth; and Z-stack of 7 slices with a 0.43 mm interval. Images of EGFP-CLSTN1- and ICAM5-mCherry- positive neurons were captured every second for 5 min per cell under confocal microscopy.
EGFP-CLSTN1- and ICAM5-mCherry- particle mobilities were measured by quantifying the lines in the kymographs. Kymographs were generated with the Multi Kymograph plug-in of the ImageJ software according to the instructions. Each line represents one vesicle. Vertical lines represent stationary vesicles. Oblique lines or curves to the left represent retrograde movements, and lines to the right indicate anterograde transports.

Neutrophils were cocultured with FITC-conjugated miR-let-7b mimics in a six-well plate for 30 minutes and were then centrifuged at 250 ×g for five minutes. Cells were then fixed with 4% formaldehyde for 20 minutes and resuspended in 200 μL deionized H₂O. A volume of five μL cell suspension was added to the gelatin-coated slide and smeared with a pipette tip. Samples were surrounded with a hydrophobic barrier using a Super Pap Pen (XLPCC, Japan, XL2001). Samples were blocked in a blocking buffer (Beyotime, China, #P0102) for 45 minutes. Slides were then incubated with 10 μg/mL of TLR4 primary antibodies (R&D Systems, USA, #AF1478) and normal goat IgG control (R&D systems, #AB-108-C) overnight at 2–8°C. Slides were washed two times using 1% PBS and then incubated with secondary antibodies (1:500, Absin, China, #abs20027) for one hour. Slides were then washed as described previously. DAPI counterstain was added, incubated two–five minutes at room temperature and coverslips were mounted. The TLR4-miR-let-7b localization was visualized using the inverted FV1000-IX81 microscope (Olympus, Japan). Images were captured at 100× and 180× objectives using the FV10-ASW software, v01.01.

After treatment with HiLyte-Aβ₄₂, the media were removed and cells were incubated with pre-warmed (37°C) solution containing mitotracker probe (250 nM, Invitrogen) for 45 minutes under growth condition. After washing twice with PBS, the cells were fixed with PBS containing 4% paraformaldehyde. The fixed cells were further incubated with 4', 6-diamidino-2-phenylindole (DAPI, 1 μg/ml) to label nuclei and rinsed three times with PBS. The confocal images were obtained with Olympus FV1000-IX81 microscope, using the vendor-provided software (OLYMPUS FLUOVIEW Ver.2.1b Viewer). Fluorescence intensity was quantified with Image J software and presented as the percentage of HiLyte-Aβ₄₂-treated group.

Cells were cultured in eight-well Lab-Tek II chambers at 0.15 × 10⁶ cells/well and differentiated into macrophages. Then, macrophages were infected with mycobacteria (MOI 1:10), treated with 200 ng of rBPI, and incubated for 1 h at 37 °C in 5% CO₂. After discarding non-phagocytosed bacteria, the cells were incubated for 24 h with the same amount of rBPI. Uninfected cells treated with rBPI were included. The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and incubated with an anti-hBPI mouse antibody (R&D Systems) and anti-mouse coupled with Alexa Fluor 488 rabbit antibody (Thermo Fisher Scientific, Waltham, MA, USA). Counter-staining with Hoechst (Enzo Life Sciences, Farmingdale, NY, USA) was used to detect nuclei. Cells were visualized under a fluorescence AxioScope.A1 microscope (Carl Zeiss, Oberkochen, DEU), and images were acquired and analyzed with the ZEN Pro software v. 2012 (blue edition) (Carl Zeiss). We analyzed the infected MDM and THP-M cell preparations in a confocal microscope to confirm the intracellular localization of mycobacteria. Images were acquired in an Olympus FV1000-IX81 microscope with the FV10-ASW v.4 software and analyzed with the Fiji software v. 1.54i [27 (link)]. Orthogonal projections of confocal sections showed bacteria inside the cell (Figure S1).