Anti cd3ε percpcy5

Spleen or BAL from IAV-infected mice at day 10 were processed to single cell suspensions, red blood cells were lysed and the remaining leukocytes stained. We used PE-labeled MHCI tetramers (D^bNP₃₆₆ or K^bPB1₇₀₃) and APC-labeled MHCI tetramers (D^bPA₂₂₄ or D^bPB1-F2₆₂) (University of Melbourne Tetramer Facility), stained with Fixable Live/Dead AquaBlue viability dye (Life Technologies), blocked with anti- CD16/32 mAb (clone 2.4G2), and stained with anti-CD3ε-PerCPCy5.5 (BD Pharmingen; clone 145-2C11), anti-CD8α-PacBlue (BD Pharmingen; clone 53–6.7) and anti-CD4-AF700 (BD Pharmingen; clone RMA4-5). Cells were acquired on a FACS Canto II flow cytometer (BD Biosciences), and data were analyzed by using FlowJo software (Treestar).

Single cell suspension of the spleen from C57BL/6 mouse were prepared by gentle mashing and passing through a 70 μm mesh Cellstrainer™ (Falcon^®). Erythrocytes were removed by incubating the cell pellet for 5 min in lysis buffer at 4 °C. Splenocytes were seeded into 96-well plates with 1.0 × 10⁶ cells/well and incubated with DiI-labeled liposomes (0.1 mM) in with FACS buffer (PBS containing 2% FBS) for 4 h. After washing with FACS buffer, the cells were incubated with 5 μg/mL CD16/CD32 monoclonal antibody (eBioscience) for 20 min at 4 °C to block Fc receptors, and then washed twice. The cells were stained with anti-F4/80-FITC (BioLegend, BM8), anti-CD3ε-PerCP-Cy5.5 (BD Bioscience, 145-2C11) and anti-CD11c-PE/Cy7 (BioLegend, N418) antibodies (each diluted 1:200 in FACS buffer) for 20 min at 4 °C. After washing twice, DiI-fluorescence in CD3ε⁺ cells (T lymphocytes), F4/80⁺ cells (macrophages), and CD11c⁺ cells (dendritic cells) was analyzed via a flow cytometric analysis. Cellular autofluorescence was subtracted from each data.