Pe conjugated anti siglec f

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 APC-conjugated anti-CD11c (BioLegend), PE-Cy7-conjugated anti-CD11b (BD Biosciences), FITC-conjugated anti-Ly6G (clone 1A8, BD Biosciences), PerCp-Cy5.5- conjugated anti-Ly6C (eBiosciences), and PE-conjugated anti-Siglec-F (BioLegend) mAbs for myeloid cell analysis. In some experiments, cells were stained with PE-conjugated anti-active caspase 3 using a commercially available kit from BD Biosciences before surface marker staining. To differentiate early-stage apoptotic cells from the late-stage apoptotic and necrotic cells, BALF cells were stained with Fixable Viability Dye (FVD) eFluor® 780 and Annexin V PerCP-eFluor® 710 (eBiosciences), using BUV395-conjugated anti-CD11b (BD Biosciences), FITC- conjugated anti-Ly6C (BD Biosciences), BV421-conjugated anti-Ly6G (clone 1A8), PE-conjugated anti-Siglec-F and APC-conjugated anti-CD11c mAb for cell surface markers. The stained cells were analyzed on a BD LSRII-green using BD FACSDiva and FlowJo software analysis.