Annexin 5 fitc detection kit

Apoptotic cells were assessed using the Annexin V-FITC Detection Kit (Vazyme, China) according to the protocols of the manufacturer. After pre-cooling, 2–3 mm of flesh under the exocarp was cut into 2 mm × 2 mm × 3 mm cuboids on ice, embedded in optimal cutting temperature (OCT), and placed on a quick-freezing rack. A 25 μm-thick sheet was cut using a freezing microtome (Model: HM525, LEICA, Germany). The cells were washed two times with cold PBS and suspended in the binding buffer. The samples were stained with 10 μl of Annexin V-FITC and 10 μl of PI for 20 min at room temperature (25°C) in the dark. The apoptotic index was immediately determined using a confocal laser scanning microscope (TCS SP5 II, Leica, Germany).

Hemocytes were harvested from oysters at 72 h post dsRNA injection. The apoptosis rate was detected by using the Annexin V-FITC detection kit (Vazyme, China) according to the manufacturer’s instruction. In the reaction system, 100 μL of hemocytes, diluted with 100 μL of binding buffer, was incubated with 5 μL of Annexin V-FITC and 5 μL of propidium iodide for 10 min to mark early apoptotic cells and late-apoptotic or necrotic cells. After recollection and re-suspension, the hemocytes were immediately subjected to flow cytometer (Guava, United States) for apoptosis rate detection (10,000 events countered).