2.8. Flow Cytometry

Endometrium tissue was harvested by scraping out the endometrium as described above. Tissue was collected in 4.8 ml of PBS in disposable aseptic culture dishes and then collected. To digest the tissue, 0.6 ml hyaluronidase (1 mg/ml) and collagenase IV (1 mg/ml) were added and incubated for 2 h in a constant temperature shock box (37°C) [19]. Cells were filtered through a 70 μm cell strainer and centrifuged for 5 minutes at 1500 rpm. The pellet was resuspended in 1 ml of sample diluent and carefully transferred to 10 ml centrifuge tubes which contained 3 ml of lymphocyte separation buffer beforehand. After centrifugation for 30 minutes at 2000 rpm and 18–28°C, cells present in the lymphocyte layer were carefully aspirated, washed with 3 ml washing solution, and then centrifuged for 10 minutes at 2000 rpm. The pellet was resuspended in 90 μl PBS, and 10 μl of diluted OX-62-FITC antibody (10 μg/ml, 1 : 10) was added and incubated in the dark for 30 minutes at 4°C. After washing with 1 ml of PBS and centrifugation for 5 minutes at 1500 rpm, the pellet was resuspended in 100 μl PBS and immediately used for FCM. Data was collected and analyzed with CellQuest and CellQuest Pro software.