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.