Anti annexin 5 fitc apoptosis detection kit

In order to evaluate the protective effect of DEFB118 against E. coli K88, IPEC-J2 cells were grown to ~60% confluence at 37°C in a CO₂ incubator (5% v/v), then incubated with DEFB118 for 12 h (DEFB118, DEFB118+E. coli K88). Cells were challenged with 1 × 10⁶ CFU/well E. coli K88 for 2.5 h (DEFB118+E. coli K88, E. coli K88); control cells were cultured in a culture medium of 2% serum (without any antibiotics) without any treatment. Treated cells were harvested and labeled with an anti-Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA). Floating cells were collected; then, attached cells were washed with 0.01 M PBS and trypsinized for 2 min. Finally, trypsinized cells and floating cells were added together to centrifugate at 350 g for 10 min, then stained with Annexin V-FITC and propidium iodide (PI). The intensity of the markers was examined by flow cytometry (FACSCanto II, BD Biosciences, USA). All flow cytometric data were analyzed using FlowJo software (BD Biosciences, USA).

The proportion of apoptotic cells in IPEC-J2 cells was determined by flow cytometry (CytoFlex, Beckman Coulter, Inc., Brea, CA, USA) using PE Annexin V Apoptosis Detection Kit I (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA). When IPEC-J2 cells were grown to ~60% confluence at 37 °C in a CO₂ incubator (5% v/v), they were incubated with PNKL for 24 h (PNKL, PNKL +E. coli K88). Cells were challenged with 1 × 10⁶ CFU/well E. coli K88 for 2.5 h (PNKL+E. coli K88, E. coli K88) before sample collection, and control cells were cultured in a culture medium without any treatment. Treated cells were harvested and labeled with an anti-Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA). Floating cells were collected, then attached cells were washed with 0.01 M PBS and digested with trypsin for 2 min. Finally, the digested cells and floating cells were added together to centrifugate at 350× g for 10 min, then the cells were stained with 2 μL of Annexin-V FITC fluorescent dye at 4 °C in the dark. After 10 min, 2 μL of PI staining was added for 5 min at 4 °C in the dark. Finally, detection of apoptotic cells was completed within 1 h after the addition of 400 μL Annexin V binding buffer (1×).