Attune nxt acoustic focusing cytometer

A549 cells were seeded in a 24-well plate, at a density of 1 × 10⁵ cells per well, the night before the experiment. The uptake of BSA nanoparticles was tested in cells with 50–60% cellular confluency. The medium was removed and FITC-BSA, pre-dispersed in 0.5 ml RPMI-1640, was added at increasing concentrations (100 μg/ml, 150 μg/ml, and 200 μg/ml). Untreated cells were used as blank control, and every concentration was tested in triplicates. After 3 h of incubation at 37°C or 4°C, the cells were washed in 500 μl PBS, and detached from the well plate with 200 μl of 0.05% trypsin solution. The trypsin was neutralized with 400 μl of RPMI-1640, and cells were centrifuged at 450 g for 5 min. After removing the supernatant, the cell pellets were resuspended in 500 μl of FACS buffer (BD Biosciences, Franklin Lakes, New Jersey, USA) and uptake efficiency was assessed by flow cytometry using the Attune™ NxT Acoustic Focusing Cytometer.
Similar experiments were performed using BSA nanoparticles encapsulating YOYO-1 labelled DNA, pDNA or siRNA labelled with Alexa 350. For uptake experiments, BSA nanoparticles were used at a final bulk DNA concentration of 250 ng and 500 ng, for pDNA at 250 ng, 500 ng, 1 μg, 1.5 μg and 2 μg, and for siRNA experiments with 30 nM and 50 nM siRNA, taking into account the loading efficiencies of each formulation.