Flow Cytometry for Cell Characterization

Antibody staining for cell surface markers and flow cytometric analysis of the staining and EGFP signals were previously described^{24 (link)}. Intracellular Foxp3 transcription factor staining was performed as described^{23 (link)}. For the intracellular cytokine staining, cells were stimulated in culture with PMA/inomycin for 2–4 hours in presence of brefeldin A first and then stained similarly as for the intracellular transcription factor staining. Cells stained with a single antibody were used to calibrate signal output and compensation. Staining with isotype control antibodies was used to set gating for corresponding antigen-specific antibody staining. Flow cytometric analyses were performed on FC500 (Beckman Coulter) or BD Fortessa LSRII (BD Biosciences, San Jose, CA). Data were analyzed with FlowJo software (TreeStar, Ashland, OR). Cell gating is indicated in figures. Cell sorting was performed using BD Cytopeia’s Influx (BD Biosciences).