Alexa fluor 647 anti mouse cd8α

Single cell suspensions were pretreated with mouse Fc-Block™ (BD Biosciences/ Pharmingen) and incubated with fluorophore-labeled antibodies or appropriate isotype controls. Acquisitions were performed using a BD FACSCanto™ II (BD Bioscience). For intracellular staining of cytokines BD Cytofix/Cytoperm (BD Biosciences) was used. Analysis was done using BD FACSDiva™ and FlowJo (Treestar INC.) software. Following antibodies were used: Phycoerythrin-(PE), allophycocyanin-(APC) and PE/Cy7 anti-human FcεRIα mAb CRA1 (clone AER-37, eBioscience or BioLegend), PE-anti-mouse IgE (RME-1, Biolegend), APC-anti-CD11c, Alexa Fluor® 647 anti-mouse CD8α and Alexa Fluor® 647 anti-mouse CD4 from Biolegend (San Diego, CA), PE-anti-mouse CD86 (BD Pharmingen), APC-anti- CD117 (Biolegend), and PE- anti-mouse FcεRIα mAb (Mar1, Biolegend). Intracellular cytokine stain included PE anti-mouse IFN-γ (clone XMG1.2, Biolegend) and APC anti-mouse IL-4 (clone 11B11, eBioscience). Anti-human CD1c (BDCA-1, clone AD5-8E7, Miltenyi Biotech), APC-anti-human CD19 (clone HIB19, BioLegend), PE/Cy7 anti- human IgE (clone MHE-18, BioLegend), PE anti-human CD203 (Biolegend), Alexa Fluor® anti human CD83 (clone HB15e), PE anti-human CD80 (Biolegend) and APC anti-human CD1a (BD Pharmingen).