Isolation of Cervical-Uterine Cell Populations

Tissues obtained from hysterectomies included endometrium (EM), endocervix (CX) and ectocervix (ECX) and were transferred to the laboratory immediately after surgery and processed as previously described (3 (link), 6 (link), 15 (link), 17 (link), 18 (link)). Vaginal tissues were not available. Tissues were rinsed with HBSS and minced under sterile conditions into 1–2 mm fragments and digested using an enzyme mixture containing 0.05% collagenase type IV (Sigma-Aldrich, St. Louis, MO) and 0.01% DNAse (Worthington Biochemical, Lakewood, NJ) for 1h at 37°C. Type IV collagenase was selected based on preliminary studies to ensure non-cleavage of surface markers (3 (link), 15 (link)). After digestion, cells were dispersed through a 250 μm mesh screen (Small Parts, Miami Lakes, FL) and filtered through a 20 μm mesh screen (Small Parts) to separate epithelial cells from stromal cells. Stromal cells were then washed and counted and dead cells removed using the Dead cell removal kit (Miltenyi Biotec, Auburn, CA) according to manufacturer instructions to obtain a mixed cell suspension for flow cytometric analysis, degranulation assays and further cell purification.