Cd8 apc cy7 53 6

Mice were injected intraperitoneally with 200 μl 3% thioglycolate medium, killed after 3.5 hours and their peritonea washed with 9 ml PBS, 5 mM EDTA. The recovered cells were resuspended in 0.2 ml HBSS, 5 mM EDTA and stained for 1 hour at 4°C for flow cytometry [antibodies used were CD11b-PE (M1/70, eBioscience), Ly6G BV510 (1A8 BD Biosciences), Ly6C-BV605 (AL-21, BD Biosciences), F4/80 BV711 (T45-2342 BD Biosciences), CD4 BV786 (RM4-5 BD Biosciences), CD8-APCCy7 (53-6.7 BD Biosciences), CD19-PE-Cy7 (1D3 BD Biosciences), CD45-PerCP-Cy5.5 (30-F11, BD Biosciences)].

For determining T cell-derived cytokine levels, intracellular cytokine staining was performed as described (56 (link)). Briefly, single-cell suspensions from the lungs were stimulated with 2 μg of HA for 5 hr at 37°C. Cells were then incubated with GolgiStop (BD Biosciences) for an additional 5 hr. After wash, cells were incubated with Fc block and then stained with fluorescently-conjugated Abs for T cell surface markers, CD3 PE (145-2C11, BD Biosciences), CD4 APC-Cy7 (RM4-5, BD Biosciences), and CD8 APC-Cy7 (53-6.7, BD Biosciences). Cells were fixed and permeabilized by incubating with BD CytoFix/Perm. After FACS buffer wash, cells were stained for cytokines IFN-γ APC (XMG1.2, BD Biosciences) and TNF-α Alexa Fluor 488 (MP6-XT22, BD Biosciences). All block, incubation, and permeabilization steps were performed for 20 min at 4°C. After wash, cells were suspended in 2% paraformaldehyde and analyzed by flow cytometry as described above.

For intravenous in vivo labeling of circulating T cells, mice were intravenously injected with 3 μg of PerCP-Cy5.5-conjugated anti-mouse CD3e Ab (145-2C11, BD Biosciences). After 10 min, lungs were perfused with 3 ml of PBS, and the single-cell suspensions were made as described above. Fc block (anti-mouse CD16/CD32, BD Biosciences, 1 μg/sample) was added to the lung and spleen cell samples and incubated for 30 min at 4°C. After washing with FACS buffer, cells were incubated with fluorescently-conjugated Abs to stain for T cell markers, CD3 PE (145-2C11, BD Biosciences), CD4 APC-Cy7 (RM4-5, BD Biosciences), CD8 APC-Cy7 (53-6.7, BD Biosciences), CD69 APC (H1.2F3, BD Biosciences), CD11a FITC (2D7, BD Biosciences), and CD103 FITC (M290, BD Biosciences), for 1 hr at 4°C. Isotype control Abs were included in each experiment. After washing, cells were suspended in 2% paraformaldehyde and analyzed using a FACSAria cytometer (BD Biosciences) and FlowJo software (Tree Star).