Annexin 5 fluorescein isothiocyanate and propidium iodide

At 12, 24, 48, 72, and 96 h post-infection (hpi), HD11 cell supernatants were collected to test the viral load of ALV-J or CIAV and the concentrations of cytokines. The ALV-J and CIAV viral load were quantified by using the published qPCR method [16 (link)] and the qPCR method as described above, respectively. The concentrations of IL-6, IL-10, IFN-α, and IFN-γ in the cell supernatants were measured through ELISA as previously described [16 (link)]. At 12, 24, and 96 hpi, HD11 cells were collected to test the apoptosis using a Dead Cell Apoptosis Kit with annexin V-fluorescein isothiocyanate and propidium iodide (Beyotime). The percentage of apoptotic cells was quantitated by using a fluorescence-activated cell-sorting (FACS) BD AccuriC6 cell sorter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

MPC‐83 cells (5 × 10⁵/well) were cultured in 24‐well plates in triplicates and stimulated with 10 nmol/L caerulein for 8 h. After washing with phosphate‐buffered saline for three times, cells were resuspended and stained with Annexin V‐fluorescein isothiocyanate and propidium iodide (Beyotime) for 15 min. The cells were then estimated by a flow cytometer (BD) and apoptosis rate was defined as the sum of the percentage of the early and late apoptosis.