Cytoexpert analysis software

A FITC Annexin-V apoptosis detection kit (BD Pharmingen, San Diego, CA, USA) was used to assess apoptosis, according to the manufacturer’s instructions. Briefly, 10⁵ cells were gently re-suspended in 100 µL of binding buffer and incubated for 15 min at room temperature in the dark with 5 µL of Annexin-V-FITC and 5 µL of Propidium Iodide (PI). After the addition of 200 µL of binding buffer, samples were analyzed with a Cytoflex flow cytometer with the FL1 and FL3 detector in a log mode, using the Cytoexpert analysis software (both from Beckmann Coulter). For each sample, 10,000 events were collected. Viable cells are Annexin-V^neg/PI^neg (unlabelled), necrotic cells are Annexin-V^neg/PI^pos, while early and late apoptotic cells are Annexin-V^pos and PI^neg and PI^pos, respectively.

A FITC Annexin-V apoptosis detection kit (BD Pharmingen, San Diego, CA, USA) was used to detect apoptosis, following the manufacturer’s instructions. Briefly, 10⁵ cells were gently re-suspended in 100 µL of binding buffer and incubated for 15 min at room temperature in the dark with 5 µL of Annexin-V-FITC and 5 µL of Propidium Iodide (PI). After the addition of 200 µL of binding buffer, samples were analyzed with a Cytoflex flow cytometer with the FL1 and FL3 detector in a log mode, using the Cytoexpert analysis software (both from Beckmann Coulter). For each sample, 10,000 events were collected.

To assess apoptosis, a FITC Annexin-V apoptosis detection kit (BD Pharmingen, San Diego, CA, United States) was used following the manufacturer’s instructions. The day before PS-NP treatment, cells were seeded at a density of 500,000/well and grown under standard culture conditions in 6-well plates. After 24 h, 10, 100, and 500 μg/mL of PS-NPs were added to cells, which were further incubated. After 96 h, 10⁵ cells were gently re-suspended in 100 μL of binding buffer and incubated for 15 min at room temperature in the dark with 5 μL of Annexin-V-FITC and 5 μL of Propidium Iodide (PI). After the addition of 200 μL of binding buffer, samples were analyzed with a Cytoflex flow cytometer with the FL1 and FL3 detector in a log mode, using the Cytoexpert analysis software (both from Beckmann Coulter, Milan, Italy). For each sample, at least 5,000 events were collected. From the same samples, the dot plots of the autofluorescence parameters (forward and side scatter) were analyzed for changes in cell size and cellular content.