Analytical Flow Cytometry of Immune Cells

All analytical flow cytometry was performed on a BD LSR II flow cytometer. Dead cells were identified by DAPI (4’,6-diamidino-2-phenylindole) and excluded from analysis where appropriate. Primary antibodies specific for the following markers were directly conjugated to FITC, PE, APC, PerCP-Cy5.5, eFluor-780, eFluor-450, or PE-Cy7 and purchased from eBioscience (CD3, CD4, CD8α, CD11b, CD16/32, CD34, CD45.1, CD45.2, CD45R(B220), CD49b(DX5), FcεR1 and Gr1) or from BD Biosciences (c-kit, IL-5Rα, Siglec F, CCR3, and Annexin V). For intracellular staining, surface-labeled cells were fixed in isotonic 3% paraformaldehyde and incubated with affinity-purified anti-CF (Hamilton et al., 2008 (link)) in Fix & Perm (Invitrogen) according to product instructions. Staining was detected with highly cross-adsorbed donkey anti-rabbit-FITC (Jackson Immunochemicals, Stratech, UK). For cell sorting experiments, eosinophil populations were sorted on a BD Influx flow cytometer.