To image the in vitro release of nanoparticles from neutrophils to 4T1 cells due to the formation of NETs in the presence of phorbol myristate acetate (PMA), DiD-labeled neutrophils encapsulated with MM-LPs-DiO (neutrophils-DiD/MM-LPs-DiO) were co-cultured with 4T1 cells in medium with 100 nmol/L PMA and observed under ImageXpress Micro system (Molecular Devices). Briefly, 4T1 cells (1 × 104 cells) were seeded in a 96-well plate. After overnight incubation, neutrophil-DiD/MM-LPs-DiO was added to tumor cells and incubated with or without PMA (100 nmol/L) for 8 h. Tumor cells were then washed with PBS slightly to remove suspended neutrophils and uningested nanoparticles followed by staining with DAPI for 30 min to localize cell nuclei.
We also verified this phenomenon in vivo. Mice were first i.v. injected or not with LPs-R848 (5 μg of R848 per mouse). One hour later, neutrophils-DiD/MM-LPs-DiO (5 × 106 cells) were i.v. injected. After 20 h, mice were sacrificed, and tumors were digested to analyze DiO signals in transferred neutrophils and tumor cells using flow cytometry (BD). Moreover, tumors, livers and spleens in selected groups were rapidly made into frozen slices for further observation. Upon staining with DAPI to mark cell nuclei, the distribution of fluorescence signals of DiD and DiO was visualized using the slide scanning system (Olympus).
We also verified this phenomenon in vivo. Mice were first i.v. injected or not with LPs-R848 (5 μg of R848 per mouse). One hour later, neutrophils-DiD/MM-LPs-DiO (5 × 106 cells) were i.v. injected. After 20 h, mice were sacrificed, and tumors were digested to analyze DiO signals in transferred neutrophils and tumor cells using flow cytometry (BD). Moreover, tumors, livers and spleens in selected groups were rapidly made into frozen slices for further observation. Upon staining with DAPI to mark cell nuclei, the distribution of fluorescence signals of DiD and DiO was visualized using the slide scanning system (Olympus).