Cd11b pe m1 70

Fluorochrome-conjugated monoclonal antibodies (mAbs), including PE-CD11b (M1/70, 101207, 1:400), PerCP/Cy5.5-IL-17A (TC11-18H10.1, 506919, 1:100), PE/Cy5-Gr-1 (RB6-8C5, 108409, 1:600), APC-CD4 (GK1.5, 100412, 1:400), FITC-TNF-α (MP6-XT22, 506304, 1:100), and APC/Fire750-CD4 (GK1.5, 100460, 1:100) were purchased from BioLegend (San Diego, CA), while PE-IL-17A (eBio17B7, 12-7177-81, 1:100) was purchased from eBioscience (San Diego, CA). Anti-mouse IL-17A neutralizing monoclonal antibody (17F3, BP0173) was purchased from BioXCell (West Lebanon, NH). Recombinant mouse IL-1β, IL-6, IL-17A, and TNF-α were purchased from BioLegend. Mouse IL-17A ELISA kit was purchased from BioLegend. Mouse IL-17A ELISPOT kit was purchased from R & D Systems (Minneapolis, MN). Native chicken and bovine collagen type II were purchased from Sigma-Aldrich (St. Louis, MO) and Chondrex (Redmond, WA), respectively.

Extracted tumors were minced completely (1–3 mm cubes) by using scalpels in two drops of dissociation solution (1 mg/ml collagenase D (11088858001 Roche, Indianapolis, IN) and 1 mg/ml DNase I (10104159001 Roche) in RPMI 1640) on ice. Tumor pieces were further digested for 45 min with dissociation solution (about 1 g tumor in 10 ml dissociation solution) at 37 °C water bath with manually periodical shaking. The digested tissue suspension was aspirated into a 20 ml syringe with 14 g cannula attached and clumps were triturated 15 times and filtered through 40 um strainer to get the single cell suspension  followed by staining with fluorescent antibodies from Biolegend: PE CD4 (GK1.5, 100407), Brilliant Violet 605 CD8a (53–6.7, 100744), PE CD11b (M1/70, 101207), Alexa Fluor 700 F4/80 (BM8, 123130), Alexa Fluor 488 CD3 (17A2, 100210), APC MHC II (M5/114.15.2, 107613). Stained cells were analyzed using BD LSRFortessa X-20 flow cytometer. Data were collected with BD FACSDiva v 8.0.1 software and were analyzed using FlowJo v 10 software.

A total of 10⁵ PFU of MA-eGFP-PR8 was intranasally inoculated into B6 mice. To label lung macrophages, 50 μl of PE-CD11b (M1/70, BioLegend, San Diego, CA) was injected intravenously to the mice at day 3 p.i. Thirty minutes after the antibody injection, the lungs of the mice were harvested. The kinetics of eGFP- and PE-positive cells in the lungs was imaged with a two-photon laser microscope (LSM 710 NLO, Carl Zeiss, Oberkochen, Germany). During the analysis, the lungs were maintained in complete medium (RPMI 1640 with 10% fetal calf serum) in a humid chamber (37 °C, 5% CO₂). The data were processed with LSM software Zen 2009 (Carl Zeiss). For three-dimensional imaging of HPAI virus-infected lung tissues, B6 mice were intranasally inoculated with 10⁵ PFU of MA-Venus-HPAI virus and MA-Venus-PR8. The lung tissues were collected from the mice at day 1 and 2 p.i., and treated with SCALEVIEW-A2 solution (Olympus) to make tissues transparent as described above. Three-dimensional images of lung tissues were obtained from a two-photon laser microscope (LSM 710 NLO).