Anti gr1 pe cy7

Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO). Anti-coxsackievirus B3 Antibody (MAB948) was purchased from EMD Millipore (Jaffrey, NH). Anti-F4/80 and anti-NFκB p65 were from Abcam (Cambridge, MA). Anti-ICAM-1 and anti-CCR2-PE antibodies were from R&D Systems (Minneapolis MN). Anti-CD16/32 Fc Block, anti-CD11b-APC/CY7, and anti-VLA-4-FITC were from Biolegend. Anti-Gr1-PE-Cy7 was from BD bioscience (San Jose, CA). Alexa Fluor 488, 594 or 633 labeled secondary antibodies were from Life Technologies (Carlsbad, California). The viability dye Ghost violet 510 was from TonBo. For real-time PCR Power SYBR Green RNA-to-Ct^TM 1-Step kit from Thermal Fisher was used.

On the indicated days after influenza virus infection, mice were terminally sedated with nembutal, and the lungs were removed and treated with collagenase and DNase. The lungs were subsequently forced through a 70 μM filter to produce single-cell suspensions. Erythrocytes were removed by lysis in NH₄Cl red blood cell lysis buffer. The cells were incubated with anti-mouse CD16/CD32 antibody (FcBlock, BD) to avoid nonspecific immunostaining of immune cells and stained with anti-B220-Alexa Fluor 700, anti-CD11c-APC, anti-CD11b-APC-Cy7, anti-CD45-PerCP, anti-GR1-PE-Cy7, anti-CD3e-PE and anti-CD49b (DX5) V450 (all from BD) for 30 min. The number of GFP positive lung cells was determined on an LSR-II flow cytometer (BD, San Jose, CA) by analyzing surface expression of CD45, CD3e, B220, CD11b, CD11c, GR1, and DX5 using FACSDiva (BD) and FlowJo software (Treestar). The gating strategy used to define the different cell subsets are presented in S1 File.

Blood and spleen samples from infected and uninfected mice and single-cell suspensions were prepared. Peripheral blood cells were isolated by Percoll gradient (Sigma-Aldrich, Brazil). Spleens were homogenized and passed through a nylon mesh of 70 μm to create a single-cell suspension. Cells were stained with Trypan blue (Sigma-Aldrich, Brazil) and assessed for viability in a haemocytometer. Cells (10⁶) were washed, resuspended in flow cytometry buffer (2% foetal calf serum (Invitrogen, Brazil) in phosphate buffered saline-PBS (Sigma-Aldrich, Brazil)), and stained with directly conjugated monoclonal antibodies (BD Biosciences or eBiosciences, Brazil): anti-F4/80 FITC, anti-IAb PE, anti-Ly6G PerCP, anti-GR1 Pe-Cy7, anti-CD3 PE and APC, anti-NK1.1 FITC, and anti-CD69 PECy5. Events were acquired on a BD FACSCanto (BD Biosciences-Immunocytometry Systems) and analyzed using FlowJo software (Tree Star Inc.). In order to analyze monocyte/macrophage and neutrophil populations, cells expressing CD3⁺, CD4⁺, CD8⁺, and CD19⁺ were gated out and the phenotypic F4/80, GR1, and Ly6G lineage markers were evaluated. Results are expressed as representative two-colour dot-plots as well as number of cells (×10⁶), except for IAb, expressed as mean fluorescence.