Studies on intracellular uptake of CLP nanoparticles in neutrophils
This protocol is extracted from research article:
A self-illuminating nanoparticle for inflammation imaging and cancer therapy
Sci Adv, Jan 9, 2019; DOI: 10.1126/sciadv.aat2953

Neutrophils were isolated from the peritoneal exudates of BALB/c mice after 4 hours of stimulation with thioglycollate (3.0 weight %). Cellular uptake profiles of CLP nanoparticles in neutrophils were analyzed by the fluorescence-activated cell sorting technique. Specifically, neutrophils were seeded in a 12-well plate at 5 × 105 cells per well in 1 ml of sterile Hanks’ balanced salt solution (HBSS) and incubated for 10 min. After washing with fresh HBSS, cells were incubated with 1 ml of growth medium containing 21.6 μM CLP nanoparticles for predetermined time periods. In another study, cells were incubated with various concentrations of CLP nanoparticles for 2 hours. After cells were washed three times with PBS, blew down, and collected by centrifugation, they were resuspended in PBS for analysis by flow cytometry. To observe cellular internalization behaviors by CLSM, neutrophils were treated with CLP nanoparticles at 21.6 μM for different time intervals. After washing three times with PBS, cells were fixed and stained with DAPI. CLSM observation was performed by a fluorescence microscope.

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