Fitc annexin 5 apoptosis detection

To detection apoptosis, cells were stained as described in the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, San diego) and counted by flow cytometry (BD FACs Calibur). FITC Annexin V Apoptosis Detection was perfomed as the manufacturer recommended (BD Pharmingen).

Cells were seeded in T-25 flasks and treated with IC50 conditions determined by the MTT assay after 24 hr of incubation with either free baicalin or the selected nanocapsules formulations. Control group (incubated with medium only) was also included as reference. At the end of the incubation period, they were dissociated from the plates using biotase and collected by 5 minutes centrifugation at 1200 rpm. Following the manufacturer’s instructions, cells were stained for 15 minutes at room temperature with Annexin V FITC and propidium iodide (PI) supplied within the kit (BD Pharmingen FITC Annexin V apoptosis detection). Cells were suspended in the provided binding buffer and the fluorescence signal of both PI and Annexin V FITC were simultaneously measured by flow cytometry. The excitation and emission wavelengths for PI were 493 and 636 nm respectively. For every sample, fluorescence of 10,000 cells was measured. Three measurement controls were included; (1) unstained cells, (2) cells stained with PI only, (3) cells stained with Annexin V FITC only. Data was analyzed with the same software mentioned before and the cumulative sum of both early and late apoptotic cells present in the right lower and upper quadrants of the dot plot were presented.