Annexin 5 fitc propidium iodide apoptosis detection kit

To analyze whether PirB protein induces apoptosis in hemocytes, primary shrimp hemocytes were treated with rPirB. Briefly, hemocytes collected from shrimp hemolymph were cultured at 28°C for 2 h, before being treated for 4 h at 28°C with 0.1 µM rPirB, PBS (negative control), or apoptosis inducer A (positive control) (Beyotime, Shanghai, China). Next, cells were collected and stained with Annexin V-FITC/Propidium Iodide apoptosis detection kit (Beyotime biotechnology, Shanghai, China) followed by flow cytometry analysis using an Accuri C6 plus flow cytometer (BD Bioscience, SanDiego, USA) based on 10,000 events recorded on the dot plot.
To further ascertain the ability of PirB protein to induce apoptosis in shrimp hemocytes, in vivo experiments were carried out. First, healthy laboratory acclimatized shrimp were divided into two groups (30 each). One group of shrimps was each injected at the third abdominal segment with 0.8 µM rPirB, while the other group of shrimps was each injected with an equal volume of PBS (negative control). Hemocytes collected at 0, 12, 24, 36, and 48 h post-treatment were stained with the Annexin V-FITC/Propidium Iodide apoptosis detection kit and analyzed by flow cytometry. Triplicate samples were analyzed per treatment for at least three independent experiments.

The apoptosis of treated HNPCs was evaluated using flow cytometry [26 (link)]. Prior to this evaluation, the Annexin V-FITC/Propidium Iodide Apoptosis Detection Kit (C1062M) was purchased from Beyotime (Shanghai, China), and treated HNPCs were collected and digested with Trypsin solution. Briefly, treated HNPCs were resuspended with PBS and the cell concentration was adjusted, after which cells (5 × 10⁴) were centrifuged (1000 × g) using a centrifuge (E2615, Beyotime, Shanghai, China) for 5 min. Afterward, cells were added with 195 μL AnnexinV–FITC conjugated solution, 5 μL of Annexin V-FITC solution and 10 μL of Propidium Iodide solution, followed by 15-min incubation at room temperature in the dark. Ultimately, the apoptosis of treated HNPCs was analyzed using flow cytometer (CytoFLEX, Beckman Coulter, Inc., Kraemer Boulevard Brea, California, USA) and the Kaluza C software (v. 1.1.2, Beckman Coulter, Indianapolis, Indiana, USA).

Treated cells were stained using the Annexin V-FITC/Propidium Iodide Apoptosis Detection Kit (#C1062M, Beyotime Shanghai, China) according to the manufacturer’s instructions. Images were visualized using a fluorescence microscope. Five visual fields were selected randomly for cell counting. Annexin V-positive cells were labeled as the apoptotic cells and the apoptotic rate (%) was calculated as the number of apoptotic cells/total number of cells×100%.