Twenty-four hours after P. aeruginosa PAO1 infection, WT mice were anesthetized with pentobarbital sodium, and the lungs were harvested and homogenized using a homogenizer (Changzheng Co., Chongqing, China) at 4°C. Lung tissue lysates were centrifuged, and 100 μL supernatant per well was further analyzed by enzyme-linked immunosorbent assay (ELISA). The concentrations of the cytokine IL-17A in the lungs were determined by using mouse cytokine ELISA kits (Sizhengbai, Beijing, China) according to the manufacturer's instructions.
For staining Th17 cells, lung specimens were minced, digested and then filtered to generate single-cell suspensions. Lung cells were stimulated at 37°C for 5.5 h with Cell Activation Cocktail containing Brefeldin A (BioLegend) and then labeled with a FITC-conjugated anti-mouse CD4 antibody. Before the cells were stained with a PE-conjugated anti-mouse IL-17 antibody (BioLegend), they were permeabilized with intracellular staining fixation/permeabilization buffer (BioLegend). The stained cells were analyzed using an LSRFortessa cell analyzer (BD, New York, USA). The data were analyzed with FlowJo software (TreeStar), and the percentage of Th17 cells relative to the total CD4+ T cell population was statistically quantified.
For staining Th17 cells, lung specimens were minced, digested and then filtered to generate single-cell suspensions. Lung cells were stimulated at 37°C for 5.5 h with Cell Activation Cocktail containing Brefeldin A (BioLegend) and then labeled with a FITC-conjugated anti-mouse CD4 antibody. Before the cells were stained with a PE-conjugated anti-mouse IL-17 antibody (BioLegend), they were permeabilized with intracellular staining fixation/permeabilization buffer (BioLegend). The stained cells were analyzed using an LSRFortessa cell analyzer (BD, New York, USA). The data were analyzed with FlowJo software (TreeStar), and the percentage of Th17 cells relative to the total CD4+ T cell population was statistically quantified.