At 24 h post-transfection, 1x106 MRC-5 cells or 1x106 patient-derived mononuclear lymphocytes were washed twice with precooled PBS. Subsequently, the DNA Reagent kit (cat. no. 340242; BD Biosciences) was used to determine the cell cycle distribution of cells, according to the manufacturer's protocol. Briefly, cells were incubated with 200 µl liquid A at room temperature for 10 min, then with 150 µl liquid B at room temperature for 10 min and finally with 120 µl liquid C in the dark at room temperature for 10 min. Subsequently, the cells were analyzed using a flow cytometer and ModFit software (v3.2; Verity Software House, Inc.). Each experiment was repeated three times.
To assess the ratio of IL-22+ mononuclear lymphocytes, 1x106/ml mononuclear lymphocytes were incubated with 250 µl BD Cytofix/Cytoperm permeabilization solution (BD Biosciences) at 4˚C in the dark for 20 min. The permeabilization was then stopped by addition of 1 ml PBS. Following centrifugation at 25˚C and 1,200 x g for 10 min, the cells were mixed with intranuclear factor (IL22+) antibody (1:20; cat. no. IC7821P-025; R&D Systems, Inc.) before incubation at room temperature in the dark for 30 min. Following washing and resuspension in PBS, the cells were examined by flow cytometry, and data were analyzed with FlowJo v10 software (FlowJo LLC).
To assess the ratio of IL-22+ mononuclear lymphocytes, 1x106/ml mononuclear lymphocytes were incubated with 250 µl BD Cytofix/Cytoperm permeabilization solution (BD Biosciences) at 4˚C in the dark for 20 min. The permeabilization was then stopped by addition of 1 ml PBS. Following centrifugation at 25˚C and 1,200 x g for 10 min, the cells were mixed with intranuclear factor (IL22+) antibody (1:20; cat. no. IC7821P-025; R&D Systems, Inc.) before incubation at room temperature in the dark for 30 min. Following washing and resuspension in PBS, the cells were examined by flow cytometry, and data were analyzed with FlowJo v10 software (FlowJo LLC).