Multiparametric Flow Cytometry Analysis

Il-4Rα surface expression was detected on lymph node cells by phycoerythrin (PE) anti-CD124 (IL-4Rα, M-1). Cell subpopulations were identified with Alexa Fluor 700, BD Horizon V500, BD Horizon V450, PerCP-Cy5.5, APC, APC-Cy7, Fluoroscein isothiocyanate, PE, PE-Cy7 or biotinylated monoclonal antibodies against CD3, CD4, CD19, Lineage, Gata-3, IL-4, IL-13, IFN-γ, IL-10, SiglecF, T1/ST2, ICOS. Biotin-labeled antibodies were detected by Allophycocyanin or Percpcy5.5. For staining, cells (1x 10⁶) were labeled and washed in PBS, 3%FCS and 0.1% NaN3. Between each step of staining, cells were washed extensively. For intracellular cytokine staining, cells were restimulated with a cocktail of PMA/Ionomycin/Monensin for 4–12 h at 37°C then fixed in 2% PFA, permeabilized and cytokine production was analyzed as previously described [28 (link)]. For intranuclear staining, a commercially available transcription buffer set (BD Bioscience) was used as per the manufacturer’s instructions. All antibodies were from BD Pharmingen (San Diego, CA) except where noted otherwise. Stained cells were then acquired on a LSR Fortessa machine (BD Immunocytometry system, San Jose, CA, USA) and data were analyzed using Flowjo software (Treestar, Ashland, OR, Usa).