Anti cd107a pe cy5 mab

Lysosomal–associated membrane protein-1 (LAMP-1 or CD107a), a glycosylated membrane protein primarily found in lysosomes, is a marker for degranulation of NK cells (26 (link)). Accordingly, CD107a is utilized as a cytotoxicity marker for functional assays. Therefore, anti-CD107a-PE-Cy5 mAb (Biolegend, San Jose, CA, USA) was added to the co-culture of PBMCs and human erythromyeloid leukemia cell line K562, with an effector/target (E:T) ratio of 10:1 for 4 hours at 37°C in the presence of anti-CD107a-PE/Cy5 mAb (Biolegend, San Jose, CA, USA). PBMCs alone were used as unstimulated control. Following the incubation, cells were stained with anti–CD3-Alexa Fluor700, -CD16-APC/Cy7 and CD56-BV650 mAbs (all from Biolegend, San Jose, CA, USA) for 20 minutes in the dark. To evaluate perforin and granzyme B content of PBMCs, samples were fixed and permeabilized according to the manufacturer protocol (Cytofix & Cytoperm Kit, Biolegend, San Jose, CA, USA), and then stained with intracellular anti-perforin-PerCP/Cy5.5 and -granzyme B-PE (Biolegend, San Jose, CA, USA) mAbs for 20 minutes. The samples were washed with PBS and were analyzed with NovoCyte flow cytometer running NovoExpress software (Agilent Technologies, USA).