Anti b220 alexa fluor 700

Cells were isolated from BAL, lungs, spleen, and draining lymph nodes and analyzed by flow cytometry as previously described^{55 (link)}. Cells were stained with the following antibodies at the concentration of 1:200: CD16/32 (i.e., Fc block, eBioscience), Fixable Viability Dye eFluor 506, anti-Ly6G PE-Cy7 or eFluor 450, anti-CD117 FITC, SiglecF PE, CD11b PE-Texas Red, anti-CD11c APC, anti-MHC II AF700, anti-CD49b PerCP eF710, anti-FcεRIα PECy7, anti-F4/80 APC/Cy7, anti-IL17A PerCP-Cy5.5 (Ebioscience), anti-TCRβ APC-CY7 (Biolegend), anti-CD4 Alexa Fluor 700 or eFluor 450, anti-CD8α PE-Texas Red or PerCP-Cy5.5, anti-TCRδ APC, anti-NK1.1 Allophycocyanin, anti-CD44 V500, anti-CD62L PE-Cy7, anti-CD11b Alexa Fluor 647 (BD Pharmingen), anti-B220 Alexa Fluor 700, PE-PBS57 loaded CD1d tetramer was from the National Institute of Allergy and Infectious Diseases Tetramer Facility. Purified anti-CD3 and CD28 antibodies were from BD Biosciences. In some cases, cells were stimulated with PMA and Ionomycin followed by an analysis of intracellular cytokine as previously described^{56 (link)}. Cells were analyzed using a BD FACS Aria II flow cytometer and analyzed with FlowJo software.

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.

Splenocytes from mice were labeled with antibodies to delineate cell populations and included anti-CD4-PE-Cy7, anti-CD4-Brilliant Violet 605, anti-CD4-Alexa Fluor 700, anti-CD8α-APC-eFluor 780, anti-B220-PerCP-Cy5.5, anti-B220-Alexa Fluor 700, anti-CD69-PerCp-Cy5.5, and anti-CD69-Brilliant Violet 605, purchased from either BD Bioscience, eBioscience or Biolegend. Cell viability was assessed with the dye Hoechst 33258 (1 µg/ml, Calbiochem-Behring Corp.). Cells were labeled with antibodies and Hoechst 33258 for 20–30 min on ice and washed twice as previously described [31] (link).