Cell staining for flow cytometry

Mice were euthanized on 3-days post-instillation of 2 × 10^⁶ PFU of PA-S for lungs and haemocytes processing. Briefly, 100 ul of blood was collected retroorbitally, blended with red blood cell lysis buffer, incubated for 5 min, and then centrifuged at 400 × g for 5 min for haemocytes collection. Lung tissues were dissected from mice, minced on ice into small pieces of less than 1 mm³ and then suspended in a 10 mL of digestion buffer consisting of collagenase I (1 mg/mL, Thermofisher, USA), collagenase IV (0.5 mg/mL, Thermofisher) and DNase I (40 U/mL, KeyGen biotech) in DMEM/F12 medium (Gibco, USA). The digestion buffer was incubated with frequent agitation at 37 °C for 50 min, and manual dispersion with 5 mL-pipette-tip every 5 min. Subsequently, an equal volume of DMEM/F12 supplemented with 10% FBS, 1 U/mL streptomycin and 1 U/mL penicillin (Gibco, USA) was added into single-cell suspensions, passed through a 70 µm nylon mesh filter (Corning, USA) and centrifuged at 400 × g for 5 min. All pelleted cells were resuspended into ice-cold PBS supplemented with 0.05% BSA. Throughout the preparation procedure, cells were maintained on ice whenever possible. Besides, mice on 14-days post-instillation of 2 × 10^⁶ PFU of PA-S were also euthanized for inguinal lymph nodes and spleens processing. Cells were collected from inguinal lymph nodes and spleens by grinding and passing throng a 70 µm nylon mesh filter (Corning, USA).

To investigate cell-mediated immune responses, mice were euthanized on 14-days post-instillation of 2 × 10^⁶ PFU of PA-S for isolation of lymphocytes in spleens. Isolated lymphocytes were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin/streptomycin, 1 mM pyruvate (all from Gibco, USA), 50 μM β-mercaptoethanol and 20 U/mL IL-2 (all from Sigma-Aldrich, USA). Simultaneously, RBD, S1, or S protein (5 μg/mL) was added to activate cells (1 × 10⁶ per well) followed by 72 h of incubation at 37 °C. Terminally, the cells were collected for flow cytometric analysis of IL-4 and IFN-γ cytokines levels.

For surface staining, 5 × 10⁵ cells were stained with the indicated antibodies at 4 °C. For intracellular staining, cells were then fixed using the Cytofix solution (BD Biosciences, USA), and incubated with fluorochrome-labeled antibodies specific for the mouse. Antibodies used for flow cytometry analysis included PerCP-Cy5.5 anti-CD45, FITC or Pacific Blue anti-CD11b, APC or AmCyan anti-Ly6C, PE or APC anti-Ly6G, PerCP-Cy5.5 anti-CD3, APC anti-CD4, AmCyan anti-CD8, FITC or PE anti-CD69, FITC anti-CD80, Qdot 655 anti-NK1.1, PE anti-Granzyme B, PE-Cy7 or PE anti-IFN-γ, PE anti-CD44, Qdot 705 anti-CD62L and Pacific Blue anti-IL-4 (all from BD Biosciences or eBiosciences, USA).