Phenotypic markers expressed by the NOD DNCD3 splenic cells.

Single-cell suspensions (106 cells) isolated from the spleen of 14-day old individual NOD males and females (n = 5 per group) were co-stained with CD3-Alexa Fluor700, CD4-APC, CD8-PerCP Cy5.5 conjugates, and either one of the following Ab-PE conjugates specific for CD28, CD69, CD25, iCTLA-4, DX5, TCR Vβ or TCR Vδ antigens. Aliquot cells were co-stained with CD3-Alexa Fluor700, CD4-PE, CD8-PerCP, and a CD1d tetramer unloaded or loaded with α-galactosyl ceramide-APC conjugate. Shown are the mean values ± SD for the frequency (%) and single-cell density (MFI) of each phenotypic marker expressed by DNCD3 splenocytes and mature CD4 T-cells (control) as determined by FACS (*ND, not done).