Neutrophils were seeded in a 12-well plate at 5 × 105 cells per well in 1 ml of HBSS and washed with fresh HBSS after 10 min. After incubation in 900 μl of HBSS with or without PMA (100 ng/ml) for 1 hour, 100 μl of aqueous solution containing 54 μM CLP nanoparticles was added, followed by luminescence imaging at predetermined time points. To separate intracellular and extracellular bioluminescence, 100 μl of aqueous solution containing 54 μM CLP nanoparticles was added and incubated for 30 min with neutrophils that were stimulated with PMA (100 ng/ml). Immediately after luminescence imaging, the extracellular medium was collected, and additional imaging was performed for the separated cells and culture medium containing PMA. Neutrophils stimulated with PMA and without treatment with CLP nanoparticles served as the Blank group. In another experiment, various concentrations of CLP nanoparticles were incubated with neutrophils prestimulated with PMA to examine the dose-response luminescence. Similarly, the effect of neutrophil count was investigated. In all cases, the plates were imaged using an IVIS Spectrum imaging system (exposure time = 5 min, f/stop = 1, binning = 8, FOV = 12.8 cm). In all cases, the quantitative analysis was performed using the Image Analysis Software provided by the manufacturer. For all data obtained with the Image Analysis Software, the corresponding background values were deducted.

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