Antibody staining for flow cytometry.

Single-cell suspensions of lungs were prepared by pressing the tissues through wire mesh screens and plating 1 × 10⁶ cells/well in a 96-well plate and were blocked with rat serum in fluorescence-activated cell sorter (FACS) buffer for 30 min at 4°C.

Following blocking, cells were incubated with FACS buffer containing rat anti-mouse CD8α (53-6.7; BD), rat anti-mouse CD3ε (145-2C11; BD), and HA₅₃₃ or NP₁₄₇ tetramers for 30 min at 4°C to identify IAV-specific CD8 T cells as previously described (1, 2). To identify pDC and CD8α⁺ DC populations, cells were incubated in FACS buffer containing hamster anti-mouse CD11c (HL3; BD), rat anti-mouse CD8α (53-6.7; BD), rat anti-mouse CD11b (M1/70; eBioscience), rat anti-mouse IA/IE (M5/114.15.2; eBioscience), rat anti-mouse CD45R (RA3-6B2; BioLegend), mouse anti-mouse MHC-I (34-1-2S; eBioscience), rat anti-mouse IL-15Rα (DNT15Ra; eBioscience), and rat anti-mouse CD70 (FR70; BioLegend) for 30 min at 4°C. Cells were then fixed in FACS lysis buffer (BD) per the manufacturer's instructions and resuspended in PBS.

For intracellular staining, cells were incubated in FACS buffer with saponin containing goat serum and 2.4G2 for 30 min on ice. Following blocking, cells were incubated with rabbit anti-HA (PY102) for 1 h on ice, washed 3 times, and subsequently incubated with goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC). Following incubation with secondary antibody, cells were washed 3 times and resuspended in PBS. Data were acquired on a BD FACSCanto II or a BD LSR II and analyzed with FlowJo software (TreeStar, Inc.).