**2.5.3 Isolation of cells and flow cytometric analysis**

Endometrial tissue was first digested in 2% collagenase-1 (Invitrogen, Grand Island, NY, USA) in DMEM (Himedia, Mumbai, India) for 1.5 to 2 hrs at 37°C and then centrifuged to isolate the stromal cells. Undigested glands were then treated with 0.25% trypsin-0.02% EDTA (Himedia, Mumbai, India) for 4–8min, washed with 10% FBS-DMEM. Single epithelial cells were isolated by centrifugation, as described previously. Isolated cells were washed, RBC lysed using RBC lysis solution and fixed in 2% paraformaldehyde (20 min at RT). Single cell suspension thus obtained was divided into five parts; four parts were stained with mouse anti-human αvβ<sup>3</sup> integrin, LIF (R&D Systems, Minneapolis, MN, USA), Muc-1 (Abcam, Cambridge, UK) and rat anti-human MECA-79 monoclonal antibody (Santa Cruz 1 Biotechnology, Inc., Santa Cruz, California, USA) according to instructions provided by the manufacturer in the manual. The fifth part remained unstained. Excess antibodies were washed out and the cells again incubated with fluorescein conjugated secondary goat anti-mouse and anti-rat IgG (R&D Systems, Minneapolis, MN, USA). After washing excess antibodies, the stained cells were analyzed using flow cytometer (BD FACS Calibur™, BD Biosciences, San Jose, CA, USA).
