Apc cy7 conjugated anti cd11b

BALF samples were collected by making an incision in the trachea and lavaging the lung twice with 0.8 ml PBS, pH 7.4. Total leukocyte counts were determined using a hemacytometer.
For flow cytometric analysis, BALF cells were incubated with 2.4G2 mAb against FcγRII/III, and stained with PE-Cy7-conjugated anti-CD11c (BD Biosciences), APC-Cy7-conjugated anti-CD11b (BD Biosciences), PE-conjugated Ly6B (Abcom), PCP-Cy5-conjugated anti-Ly6C (eBiosciences) and PE-Cy7 conjugated anti-Ly6G mAb (BD Biosciences) for myeloid cell analysis. APC-conjugated anti-CD11c, APC-Cy7-conjugated anti-CD11b (BD Biosciences), PE-conjugated anti-MHC II (BD Biosciences) and PCP-Cy5-conjugated anti-F4/80 (eBiosciences) were used for DC analysis. PCP-Cy5-conjugated anti-CD3 (Biolegend), APC-conjugated anti-CD4 (BD Biosciences) and APC-Cy7-conjugated anti-CD8 mAb (BD Biosciences) were used for T cell analysis. Splenocytes and BALF cells were stained with Tetramer Alexa 647-Labeled H-2D(b)/PA₂₂₄(SSLENFRAYV) and BV421-Labeled H-2D(b)/NP₃₆₆ (ASNENMETM), using FITC-conjugated anti-CD3 (BD Biosciences), APC-Cy7-conjugated anti-CD4 (BD Biosciences) and PE-conjugated anti-CD8 mAb (BD Biosciences) for cell surface markers. The stained cells were analyzed on a BD FACSCanto or BD LSRII-green using FlowJo and BD FACSDiva software analysis.

A CNS or spleen leukocyte suspension was obtained as described previously.^{49 (link)} Freshly isolated cells (10⁶) were incubated with an anti-mouse CD16/CD32 FC (eBioscience) and labeled with anti-mouse antibodies: PE anti-CD25; PE anti-CD3; PerCP-Cy5.5-conjugated anti-CD45; PerCP-Cy5.5-conjugated anti-B220; PE-Cy7-conjugated anti-CD39; PE-Cy7-conjugated anti-CD8a; PE-Cy7-conjugated anti-CD19; APC anti-CD3; APC-Cy7-conjugated anti-CD4; APC-Cy7-conjugated anti-CD11b (all from BD Pharmingen); APC anti-CD3; and PE-Cy7-conjugated anti-CD39 (both from eBioscience). To detect Foxp3, the cells were suspended in the fixation/permeabilization buffer and stained with a PE anti-Foxp3 antibody (BD Pharmingen). Cell viability was assessed with the LIVE/DEAD Fixable Green Dead Cell Stain Kit or LIVE/DEAD Fixable Red Dead Cell Stain Kit (Life Technologies Corporation). At least 10,000 events were acquired in each experiment on a FACSaria flow cytometer (BD Biosciences) and the data were analyzed using FlowJo software (v.10; Tree Star).

For surface marker staining, cells were stained in PBS supplemented with 1% FBS for 30 min on ice with the following antibodies (PerCP‐conjugated anti‐CD3, BV421‐conjugated anti‐CD4, PE‐conjugated anti‐CD8, BV570‐conjugated anti‐CD45, BV421‐conjugated anti‐F4/80, BV650‐conjugated anti‐CD86, APC‐conjugated anti‐CD206, PE‐Cy7‐conjugated anti‐CD25, FITC‐conjugated anti‐γδ TCR, APC‐Cy7‐conjugated anti‐CD11b, PE‐conjugated anti‐Ly‐6G, FITC‐conjugated anti‐CD49b, PE‐conjugated anti‐CD19, PE‐conjugated anti‐ST2 antibodies, and APC‐conjugated anti‐NKp46: BD Biosciences, San Jose, CA, USA). After fixation and permeabilization, staining with an APC‐conjugated anti‐FOXP3 antibody (eBioscience) was performed according to the manufacturer's instructions. For IL‐33 staining of primary mouse hepatocytes, cells were stained using a biotinylated anti‐IL‐33 monoclonal antibody (Enzo Life Biosciences, Raamsdonksveer, The Netherlands) and PE‐Cy7‐labeled streptavidin (BD Pharmingen, San Diego, CA, USA). All the flow cytometric data were analyzed and plotted using FlowJo software (TreeStar, Ashland, OR, USA).