Apoptosis Assay for 3D NP Cells

Twenty-four hours after exposure to each agent, the NP cell–alginate 3D composites were dissolved in 55-mM sodium citrate at 4°C until the beads released the cells, which were collected by centrifugation. The NP cells were washed twice in phosphate-buffered saline, counted (3.6 × 10⁵), and labeled with the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit II (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Both early apoptotic cells (FITC+/PI−) and late apoptotic cells (FITC+/PI +) were monitored with a flow cytometer (FACS Cant; BD biosciences, CA, USA). The present study included both early and late apoptotic cells, and the results of both groups of cells were combined in the analysis [11] (link), [16] (link), [17] (link).