Gr 1 pe

CD4-PE, CD8-PE, CD69-FITC, B220-FITC, NK1.1-PE, CD3-FITC, Gr-1-PE, CD11b-FITC, CD45-Percp and iso-type controls were purchased from BioLegend (San Diego, CA). Single-cell suspension from the liver, spleen, blood and mesenteric lymph nodes were prepared as described [24 (link)]. All cells were incubated with fluorescence-conjugated mAbs in the presence of 2.4G2 or rat sera, and fluorescence-conjugated isotype mAbs were used as background fluorescence. Flow cytometric analyses were performed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), and data were analyzed using the FlowJo or Cell-Quest software.

Cell populations from BAL were assessed using fluorescence antibody staining and flow cytometry techniques. A total of 2 × 10⁶ cells were used with viability staining performed with a Live/Dead staining kit (Invitrogen-Molecular Probes, Carlsbad, CA). The cells were washed and resuspended in Dulbecco’s PBS with 1% fetal bovine serum. Cells were divided into two equal sets containing 1 × 10⁶ cells each. Fc receptors (CD16/CD32) were blocked using mouse Fc block antibodies (BioLegend, San Diego, CA) for 10 minutes at 4°C. Diluted fluorescent antibodies specific for the different surface markers or control antibodies were added to the cell suspension and incubated for 10 minutes at 4°C. The cells were washed, formalin fixed, washed again, and resuspended in flow buffer and assayed the same day. The following monoclonal antibodies were obtained from BD Biosciences (San Jose, CA): Gr-1-PE, CD11c-PE; from BioLegend: Ly6C-FITC, F4/80-Alexa Fluor 647, F4/80-Alexa Fluor 700, A-I/b-Alexa Fluor 647, CD11b-PE-Cy7, and CD11c-APC-Cy7. Data were collected using a LSR II BD Biosciences flow cytometer and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR). Forward light scatter low Gr-1+ CD11b+ F4/80− cells were identified as neutrophils and forward light scatter high F4/80+ CD11b+ cells were identified as macrophages.