Multiparametric Analysis of Lymphocyte Subsets

Lymphocytes were isolated from spleens after organ removal and analyzed separately for each mouse by flow cytometry. Cell surface stainings were performed according to standard procedures using antibodies against CD4, CD8, CD19 and CD25 directly conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) purchased from BD Pharmingen (San Diego, CA). Anti-CD4-Tri-Color was purchased from Caltag Laboratories (Hamburg, GER). Monoclonal antibodies (mAbs) for intracellular stainings (anti-IL-4⁺, anti-IL-17⁺, anti-IFNγ⁺) conjugated to PE or allophycocyanin (APC) were purchased from BD Pharmingen. PE- or FITC-labelled mAbs to FoxP3 were from eBioscience (Vienna, AUT). Intracellular cytokine staining was performed with PE- or APC- labelled mAbs as described [41 (link)]. In brief, isolated cells were stimulated with plate-bound anti-mouse CD3e mAbs (clone 145-2C11), and anti-mouse CD28 mAbs (clone 37.51, both BD Pharmingen) at 1μg/ml for 5 hours under the presence of BD-GolgiStop Protein Transport Inhibitor (containing Monensin, BD Biosciences, San Diego, CA). All flow cytometry was performed on a FACSCanto II and analyzed using FACSDiva software (all from BD Biosciences). The main cell types were quantified as their percentage in the entire lymphocyte populations, while T-cell subsets where quantified as their percentage in CD4+ lymphocyte populations.