Annexin 5 mcherry apoptosis detection kit

EdU incorporation experiment and Annexin V staining were performed using the EdU-555 cell proliferation kit (#C0075S; Beyotime, Shanghai, China) and Annexin V-mCherry apoptosis detection kit (#C1069S, Beyotime), respectively, by following the manufacturer’s instructions. The images were captured using an Olympus CKX53 microscope.

Apoptotic cells were evaluated using the Annexin V-mCherry apoptosis detection kit (Beyotime, C1070S). The staining procedure was carried out according to the instructions of the manufacturer. In brief, the EPC cells were seeded in 24-well plates overnight and transfected with pEGFP-N1 or carp TRIF-EGFP. At 24 h post-transfection, cells were harvested, then washed with PBS, and resuspended in Annexin V-mCherry Binding Buffer. Stained cells were incubated with 5 μl of Annexin V-mCherry for 10 min at room temperature in the dark. Then, the stained cells were checked with the FACS Calibur system (BD Biosciences, USA) and evaluated with the FlowJo software (TreeStar, Ashland, OR, USA). The results were calculated from three independent replicates.

Cell death detection was performed according to the manufacturer's protocols for the Apoptosis and Necrosis Assay Kit (Beyotime, C1056) and the Annexin V–mCherry Apoptosis Detection Kit (Beyotime, C1069S). HEK293T cells were cultured in 6-well plates and transfected with the indicated plasmids. After 24 h, the cells were harvested by centrifugation at 1000× g for 5 min at 4°C. For the apoptosis and necrosis/pyroptosis assay, the cells were resuspended in 200 μl of 1× binding buffer containing 5 μl Hoechst and 5 μl PI and incubated for 20 min at room temperature. For Annexin V–mCherry apoptosis detection, 1 × 10⁵ cells were gently resuspended in 195 μl Annexin V–mCherry binding buffer and 5 μl Annexin V–mCherry and incubated at room temperature in the dark for 20 min. Finally, the cells were examined by flow cytometry (Gallios, Beckman) and analyzed by Kaluza analysis software.