The TUNEL assay was performed using the “In Situ Cell Death Detection Kit” (Roche, United States), according to the manufacturer’s instructions. Briefly, A549 cells were seeded in 6-well plates (2 × 106/well) overnight. Afterward, cells were infected with reassortant viruses at MOI = 3 for 1 h, incubated with fresh infection media for 10 h at 37°C, washed with 1× PBS, dissociated using Accutase cell dissociation reagent (Invitrogen) and transferred into a 1.5 ml Eppendorf tube. The cells were then fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) in 1x PBS (pH 7.4) overnight at 4°C, washed twice with 1x PBS and centrifuged at 6000 rpm for 5 min.
The cell pellets were permeabilized with 1% Triton X-100 in 0.1% sodium citrate on ice for 20 min. Cells were washed with 1x PBS and incubated with 50 μl reaction mixture (provided in “In Situ Cell Death Detection Kit”, Invitrogen) or the “no enzyme” control in 50 μl label solution (provided in “In Situ Cell Death Detection Kit”, Invitrogen) for 1 h in the dark. Finally, cells were washed twice with 1x PBS and fixed with 3.7% formaldehyde in 1x PBS. Fluorescence was quantified by flow cytometry (BD LSRFortessa Cell Analyzer, BD Biosciences).
The cell pellets were permeabilized with 1% Triton X-100 in 0.1% sodium citrate on ice for 20 min. Cells were washed with 1x PBS and incubated with 50 μl reaction mixture (provided in “In Situ Cell Death Detection Kit”, Invitrogen) or the “no enzyme” control in 50 μl label solution (provided in “In Situ Cell Death Detection Kit”, Invitrogen) for 1 h in the dark. Finally, cells were washed twice with 1x PBS and fixed with 3.7% formaldehyde in 1x PBS. Fluorescence was quantified by flow cytometry (BD LSRFortessa Cell Analyzer, BD Biosciences).