Multiparametric Flow Cytometry Analysis

Various fluorochrome labeled antibodies specific for CD8 (53-6.72), CD4 (RM4-5), CD3 (145-2C11), TCRVα8.3 (B21.14), TCRVβ8.1.2 (KJ16), TCRVβ4 (KT4), CD44 (IM7.8.1), CD62L (MEL-14), CD25 (7D4), CD19 (ID3), CD11c (N418), CD11b (M1/70), PDCA-1 (927), CD80 (16-10A1), CD86 (GL-1), CTLA-4 (IB8), GITR (YGITR765), GITRL (YGL386), and CD70 (FR70), were purchased from BD Bioscience (San Jose, CA, USA) or BioLegend (San Diego, CA). Anti-mouse Foxp3 (FJK-16s) was purchased from eBioscience (San Diego, CA). NRP-V7-H-2K^d Tetramer-KYNKANVFL-PE was acquired from the NIH tetramer core facility (Atlanta, GA). Anti-mouse CD25 (PC61.5) and CD4 (GK1.5) were purchased from Bioxcell (West Lebanon, NH). Dead cells were excluded by DAPI staining. Stained cells were acquired using a FACS LSRII instrument (BD Bioscience). All flow cytometric data were analyzed with FlowJo (Tree Star, Ashland, OR). In one experiment the previously described flow cytometry based assay (13 (link)) was used to compare the ability of residual myeloid APC from NOD-scid and NSG mice to support regulatory T-cell (Treg) activity. In studies assessing their proliferative capacity by flow cytometry, Tregs (Foxp3-eGFP⁺) or conventional T-cells (Tconv; Foxp3-eGFP⁻) cells were stained following the manufacturers recommendations with the Dye eFluor^®670 (eBioscience, San Diego, CA) that dilutes with each division cycle.