Percp cy5 conjugated anti cd45

Single-cell suspensions were preincubated with FcγR-specific blocking mAb (2.4G2) and washed before staining. Cells were stained with the following antibodies Percp-cy5-conjugated anti-CD45 (eBioscience), APC-conjugated anti-F4/80 (eBioscience), PE-cy7-conjugated conjugated anti-CD11c (eBioscience), FITC–conjugated anti-CD206 (BioLegend), APC-conjugated anti-CD3 (eBioscience), FITC–conjugated anti-CD4 (eBioscience); For intracellular staining, cells were permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and incubated with PE–conjugated anti-IL-4, PE–conjugated anti-IL-5, PE–conjugated anti-IFN-γ (eBioscience), rabbit anti-Ym-1 (Stem Cell Technologies) and PE–conjugated anti-rabbit IgG (eBioscience). Cells were analyzed on a LSRII (BD Biosciences) with FlowJo ver.10 software (TreeStar).

Single‐cell suspensions of lung tissue were preincubated with FcγR‐specific blocking monoclonal antibody (2.4G2) and washed before staining. To quantify Th2 cells, single cells prepared from lung tissue were stimulated with phorbol 12‐myristate 13‐acetate (100 ng/ml), ionomycin (1 μg/ml), and Golgi stop, then pooled cells from mouse per group were stained with phycoerythrin/cy5 (PE/cy5)‐conjugated anti‐CD3 antibody and fluorescein isothiocyanate (FITC)‐conjugated anti‐CD4, PE‐conjugated anti‐IL‐13, or PE‐conjugated anti‐IL‐4 antibodies. For analysis of macrophages population, cells were stained with the following antibodies: Percp‐cy5‐conjugated anti‐CD45 (eBioscience), FITC‐conjugated anti‐F4/80 (eBioscience), allophycocyanin (APC)‐cy7‐conjugated anti‐CD11c (eBioscience), goat anti‐CD206 (R&D Systems Inc.), rabbit anti‐Ym‐1 (Stem Cell Technologies), APC‐conjugated anti‐goat IgG, or PE‐conjugated antirabbit IgG (eBioscience). Cells were analyzed on an LSRII flow cytometer (BD Biosciences) using FlowJo version 10 software (TreeStar, Inc.).