For apoptosis analysis, adherent cells were detached by trypsinization and suspended in 500 μL 1 × binding buffer. Then, cells were incubated with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) in the dark for 30 min at room temperature. Within 4 h, the percentage of early and late apoptotic cells was determined by the NovoCyte flow cytometer.
Bronchoalveolar lavage fluid (BALF) from mice was obtained by washing the lungs with 0.6 mL PBS three times. After centrifugation (500 × g; 5 min), BALF supernatants were collected and stored at −80 °C. BALF cell suspensions (1 × 106 cells/100 μL) were collected and stained with FACS buffer (2% BSA with 0.01% azide in PBS). Cells were then stained with fluorescein-labeled anti-mouse mAbs purchased from eBioscience for 30 min at 4 °C: CD45-APC-eFluor780 (clone NO.30-F11), CD3-PE-Cyanine7 (clone NO. UCHT1), CD4-FITC (clone NO. RM4-5) and CD8-APC (clone NO. 53–6.7). After staining, cells were washed twice and resuspended in 300 μL FACS buffer for analysis on a NovoCyte flow cytometer.
Bronchoalveolar lavage fluid (BALF) from mice was obtained by washing the lungs with 0.6 mL PBS three times. After centrifugation (500 × g; 5 min), BALF supernatants were collected and stored at −80 °C. BALF cell suspensions (1 × 106 cells/100 μL) were collected and stained with FACS buffer (2% BSA with 0.01% azide in PBS). Cells were then stained with fluorescein-labeled anti-mouse mAbs purchased from eBioscience for 30 min at 4 °C: CD45-APC-eFluor780 (clone NO.30-F11), CD3-PE-Cyanine7 (clone NO. UCHT1), CD4-FITC (clone NO. RM4-5) and CD8-APC (clone NO. 53–6.7). After staining, cells were washed twice and resuspended in 300 μL FACS buffer for analysis on a NovoCyte flow cytometer.