Krome orange conjugated anti cd3 clone ucht

Immunophenotyping was performed as described before (15 (link)). Briefly, 100 μl heparinized blood were mixed with antibodies: Krome Orange-conjugated anti-CD3 (clone UCHT, Beckman Coulter, Brea, USA), Pacific Blue-conjugated anti-CD8 (B9.11, Beckman Coulter, Brea, USA), Allophycocyanin (APC)-conjugated anti-CD107a (clone H4A3, Beckman Coulter, Brea, USA) and Allophycocyanin (APC)-conjugated anti-CD314 (ON72, Beckman Coulter, Brea, USA). Appropriate isotype controls were used. After vortex, all tubes were incubated for 20 min in the dark at room temperature. Next 3 ml of VersaLyse™ were added in each tube and the suspension was mixed gently with vortex. Then the tubes were incubated for 12 more minutes in the dark. Thereafter the tubes were centrifugated and the supernatant was aspirated. The cell pellet was washed with 3 ml of phosphate buffered saline (PBS). This washing step was repeated and finally 300 μl PBS were added before cells were immediately analyzed with a fluorescence activated cell sorter (FACS) NAVIOS^TM from Beckman Coulter. Kaluza Analysis Software (Version 1.5, Beckman Coulter) was used for analysis of flow cytometric data.