7 amino actinomycin d 7 aad kit

Apoptotic cells were quantified using an Annexin-V phycoerythrin (PE)/7-Amino Actinomycin D (7-AAD) kit (Becton Dickinson, Franklin Lakes, NJ, USA) using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Adherent cells were harvested, washed, re-suspended in 0.5 ml PBS buffer and co-stained with 5 μl Annexin-V PE and 5 μl 7-AAD for analysis by flow cytometry. The density plots using this method illustrate four cell populations according to the fluorescence characteristics: live; early apoptotic; late apoptotic; and necrotic (or dead). In this assay, live cells would be expected to be both PE-negative and 7-AAD-negative; early apoptotic cells would be PE-positive and 7-AAD-negative; late apoptotic cells would be PE-negative and 7-AAD-positive; and necrotic (dead) cells would be both PE-positive and 7-AAD-positive.

Apoptosis was measured using the Annexin V-phycoerythrin (PE)/7-amino-actinomycin D (7-AAD) Kit (Becton Dickinson, Franklin Lakes, NJ, USA), according to the manufacturer's instructions. Briefly, collected cells were washed with cold phosphate-buffered saline and resuspended in binding buffer at a density of 1 × 10⁶ cells/ml. Cells (1 × 10⁵) were then incubated with 5 ml of Annexin V-PE and 7-AAD at 25°C for 15 min in dark before adding 400 ml of binding buffer to each tube. Apoptosis was analyzed using an ABI flow cytometer (ABI, USA) within 1 h.