Lsm900

THP‐1 cells (2 × 10⁵ cells) were seeded in a 20‐mm glass bottom dish (NEST, Wuxi, China) and differentiated into macrophages. They were incubated with pHrodo red‐ and fluorescein‐labeled 10000 MW dextran (10 µg mL⁻¹) (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h before OM (500 µg mL⁻¹) or chloroquine (30 µM) treatment for 3.5 h. Cells were then washed 3 times with PBS and imaged on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany). The fluorescence intensity of pHrodo red (ex: 565 nm; em: 585 nm) and fluorescein (ex: 488 nm; em: 525 nm) in the cells was quantified by Image J software (NIH, Bethesda, MD, USA). For each condition, at least 30 cells from 3 independent experiments were quantified to obtain the intensity ratio of fluorescein to pHrodo red.
To examine the cellular uptake of Nano‐OM, THP‐1 cell‐derived macrophages were treated with DiD (Solarbio, Beijing, China)‐labeled liposomes for localizing Nano‐OM in the cells, and stained with 25 µM DiO (Beyotime, Shanghai, China) for 20 min for labeling the cell membranes and DAPI (Beyotime, Shanghai, China) for 5 min for labeling the nucleus; the fluorescence images were acquired on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany).

THP‐1 cells (2.4 × 10⁵ cells) were seeded in a 20 mm glass bottom dish (NEST, Wuxi, China) and differentiated into macrophages as previously described. To examine the cellular uptake of M‐P12, macrophages were treated with DiD‐labelled M‐P12 (0.2 mg mL⁻¹ phospholipid and 0.77 µg mL⁻¹ DiD) overnight, and stained with DiO for 20 min and DAPI for 5 min to label the cell membrane and nucleus, respectively. The fluorescence images were acquired on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany).
To assess the endosomal acidification, macrophages were incubated with pHrodo red (10 µg mL⁻¹) and fluorescein‐labelled dextran (20 µg mL⁻¹) in the presence of nanomicelles (phospholipid: 0.2 mg mL⁻¹) or chloroquine (CHQ, 30 µm) treatment for 6 h. Cells were then washed 3 times with PBS and imaged on a confocal microscope (LSM900, Leica, Wetzlar, Hessen, Germany). The fluorescence intensities of pHrodo red (ex: 565 nm; em: 585 nm) and fluorescein (ex: 488 nm; em: 525 nm) were quantified by Image J software. For each condition, at least 60 cells from 3 independent experiments were quantified to obtain the intensity ratio of fluorescein to pHrodo red.

Immunocytochemistry of neural cultures was performed at 14–22 days in vitro (DIV). Neural cultures were fixed with 4% (w/v) PFA and 4% (w/v) sucrose in PBS for 15 min at RT and then permeabilized with 0.2% Triton X-100 in PBS for 10 min at RT. After blocking with PBS containing 0.3% BSA, cells were incubated with primary antibody containing 0.3% BSA for 60 min at RT. Primary antibodies were used at the following dilutions: anti-synapsin1 (1:1,000), anti-EndoI (1:1,000), anti-Bassoon (1:2,000), anti-Complexin 1/2 (1:500) and anti-VGLUT1 (1:1,000 for Shigeo3 and 1:2,000 for Synaptic Systems). After washing with 0.3% BSA in PBS, cells were incubated with secondary antibody containing 0.3% BSA for 60 min at RT. Secondary antibodies conjugated with fluorophore were used at 1:1,000. Finally, after extensive washing, cells were post-fixed with 4% PFA for 15 min at RT and mounted in Prolong Diamond. Specimens were visualized with Carl Zeiss LSM-900 and Leica TCS SP8 confocal fluorescence microscopes with a 63× oil-immersion (NA =1.40) and a 100× oil-immersion (NA = 1.40) objective lens, respectively. Acquired images were analyzed with Fiji and images shown in figures (Figures 7, 8A, 8B, S5, and S6A) were then smoothed with a Gaussian spatial filter (the radius of 1 pixel (200 nm) for Figures 7 and S5; σ = 1.00 for Figures 8A, 8B, and S6A).