Nk1.1 apc

Infected mouse lungs (500 SeV pfu/gram body weight) were mechanically homogenized using the gentleMACS tissue dissociator (Miltenyi). The resulting cell suspension was passed through a 70 μm mesh filter then washed once in ice-cold PBS. Cells (300,000) were incubated with Fc block for 20 min on ice then stained with fluorescent-labeled primary antibodies for 30 min on ice in the dark. CD45-PerCP, NK1.1-APC, Gr-1-FITC and CD3-Alexa Fluor 700 antibodies were purchased from BD Biosciences. The F4/80-efluor 450 was purchased from eBioscience and the CD11b-PE was purchased from Invitrogen. Cells were washed once in ice-cold staining buffer (PBS + 1% BSA and 0.1% sodium azide) then fixed with 1% formaldehyde. Total number of specific leukocyte population per lung was calculated as follows: # of specific leukocyte population = total lung cell number x %gated cells x %single cells x %CD45⁺ cells x %specific leukocyte population. Flow cytometric data were analyzed with FlowJo software (Treestar, Ashland, OR).

The heparinized blood from each animal was divided into two parts. A volume of 150 µl was designated for blood count and measured using an ABX Pentra 60 C+ hemoanalyzer (Horiba, Kyoto, Japan). The rest of the blood (200–300 µl) was lysed using EasyLyse™ (Dako, Glostrup, Denmark). The amount of cells in suspension was as determined by Turk’s solution (2% acetic acid; Sigma-Aldrich) using a hemocytometer chamber. Cells (5 × 10⁵/100 µl) were marked with two panels of monoclonal antibodies. The first panel was delineated to detect T, B, and NK lymphocytes (CD3ϵ, CD4, CD8, CD19, and NK1.1). The second panel was designed for determination of monocytes and neutrophils (CD11b, F4/80, Ly6C, and Ly6G). The following monoclonal antibodies with fluorochromes for flow cytometry analysis were purchased from BioLegend (San Diego, CA, United States): anti-mouse CD3ϵ–FITC, CD4–BV421, CD19–PE, CD11c–BV421, CD45–APC/Fire™750, F4/80–PE, and from BD Bioscience: CD8–PECy7, CD11b–BV510, Ly6C–FITC, Ly6G–PECy7, and NK1.1–APC.

For intracellular cytokine staining, cells were stimulated for 5 h in RPMI 1640 medium containing 10% fetal bovine serum and penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA) with PMA (1 μg/mL), ionomycin (1 μg/ mL), and Golgiplug (BD Biosciences, Franklin Lakes, NJ). Surface staining was performed for 20 min after incubating with FcBlock (BD Biosciences) for 5 min at 4 °C, and intracellular staining was subsequently performed using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions. A single-cell suspension was stained with anti-mouse CD45 (BV510, BD Biosciences), CD3ε (PE-cy5, Thermo Fisher Scientific), CD4 (eFluor® 450, Thermo Fisher Scientific), γδTCR (FITC, Thermo Fisher Scientific), CD8a (PE-Cy™, BD Biosciences), B220 (PE, Thermo Fisher Scientific), and NK-1.1 (APC, BD Biosciences) for surface staining. For intracellular staining, a single-cell suspension was stained with anti-mouse INF-γ (PE-Cyanine7, Thermo Fisher Scientific), IL-17A (PE, Thermo Fisher Scientific), IL-22 (APC, Thermo Fisher Scientific), and Foxp3 (APC, Thermo Fisher Scientific). Cells were analyzed using MACS Quant Analyzer with Flowlogic software (Miltenyi Biotec, Gladbach, Germany).