Detection of pNK cytotoxic granules by flow cytometry
Peripheral blood was collected before 10:00 AM during the mid-luteal period of the menstrual cycle and placed in heparinized tubes. 100 uL peripheral blood was drew and incubated with antibodies against the cell surface markers CD3 (5 μL) and CD56 (5 μL) for 15 min. The erythrocytes were lysed by 1x FACS lysis solution (3349202; BD Pharmingen). The cells were permeabilized by 1x Permeabilizing Solution 2 and the cells were incubated for 10 min. Next, the cells were incubated with antibodies against the cytotoxic granules granzyme B, perforin and granulysin (10 μL each) for 30 min. Finally, the cells were analyzed by BD FACSCanto II flow cytometer equipped with BD FACSDiva software.