Accuri c6 cflow sampler flow cytometer

Cells were plated for 3 days in 6-well plates as described in Section 2.2 and treated for different times (0.5, 2, 6, and 24 h) with 1 μg of OVA-FITC either in a free state, or associated with 10 µg of nanoparticles (1/10 w/w ratio). The cells were washed with PBS and collected using trypsin, immediately inhibited by the addition of serum. The cells were then centrifuged and resuspended with PBS just prior to analysis by flow cytometry with a BD Accuri™ C6 CFlow Sampler flow cytometer (BD Bioscience, USA). The cells were selected related to their size and complexity and the mean fluorescence intensities corresponding to the amount of particles taken up or adsorbed to the cells were reported.

The 16HBE14o-(16HBE) human bronchial epithelial cell line obtained from Dr. Gruenert D. C. (Colchester, Vermont, USA), was maintained in Dulbecco's Modified Eagle Medium (DMEM, ThermoFisher Scientific, France) supplemented with 10% heat-inactivated Fetal Calf Serum (FCS, ThermoFisher Scientific, France), 100 U/ml Penicillin, 100 mg/ml streptomycin and 1% l-glutamine at 37 °C in a humidified 5% CO₂ atmosphere. The cells were plated at a density of 7.5 × 10⁵ cells/well in 6-well plates and used once they reached the confluence, after two days. The cells were treated for different times (0, 0.5, 1, 3, and 24 h) with NPL-FITC, NPL-DiD or with free OVA-FITC or OVA-FITC formulated in NPL. The cells were then analyzed with a BD Accuri™ C6 CFlow Sampler flow cytometer (BD Bioscience, USA).