2.5. Flow Cytometry
A549 cells were seeded in 6-well plates with partitions, the experiment was set as NC group, IAV group, and IAV+ AS-IV group, which were given corresponding treatment for 24 h. The cells are then digested and collected with trypsin (GENOMBIO, Fujian, China). The ROS assay kit (Beyotime, S0033S, Shanghai, China) was used to detect the intracellular total ROS levels. Fluorescence probe DCFH-DA was diluted in serum-free medium at 1:1000 to a final concentration of 10 μ mol/L. The cells were suspended in diluted DCFH-DA at a cell concentration of 5 million/ml and incubated in a cell incubator at 37°C for 20 min. Invert and mix every 3-5 minutes so that the probe is in full contact with the cells. The cells were then washed three times with PBS to fully remove DCFH-DA that had not entered the cells. DCFH-DA was hydrolyzed by esterase in cells to produce DCFH, and DCFH was oxidized by ROS species to produce DCF with fluorescence. Finally, DCF was detected by a flow cytometer (CytoFLEX; Beckman). Because the fluorescence spectrum of DCF is very similar to that of FITC, the parameter setting of FITC was used to detect DCF in this experiment.