Cd27 o323

Cells were stained as previously described [13] (link). We used monoclonal antibodies against the following antigens (clones shown in parentheses): CD45 (HI30), CD3 (HIT3a), CD4 (OKT4), CD8 (HIT8a), CD56 (N-CAM), CD27 (O323) and CD62L (Dreg 56) (eBioscience, San Diego, CA). Antibodies and isotype-matched controls were directly conjugated to either fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin- cyanine 7 (PE-Cy7), allophycocyanin (APC), Pacific Blue, Alexa Fluor 700, or biotin, with the biotinylated antibodies developed using streptavidin-phycoerythrin-cyanine 5 (SA-PE-Cy5). We assessed viability in all experiments using a Live/Dead Fixable Near-IR Dead Cell Stain Kit for 633 nm excitation (Invitrogen, Carlsbad, CA). Immediately after staining, cells were fixed and stored in staining buffer plus 2% paraformaldehyde; tubes were then stored at 4°C in a rack wrapped in aluminum foil until analyzed.
Experiments were performed on an LSR II flow cytometer (BD Bioscience, San Jose, CA), equipped with the following lasers: 488 nm blue, 405 nm violet laser, 633 nm red HeNe laser, and a 561 nm yellow-green laser. Data were collected using FACSDiva software (BD Biosciences) with automatic compensation and were analyzed using FlowJo software (Tree Star, Ashland, OR). We collected at least 10,000 viable CD45+ events per sample in each experiment.