Anti cd45 antibody coated dynabeads

Endometrial tissue was minced and digested with phosphate-buffered saline (PBS) containing 0.3 mg/ml collagenase III (Worthington Biochemical Corporation, Freehold, NJ, United States) and 40 μg/ml deoxyribonuclease type I (Worthington Biochemical Corporation) in a shaking water bath for 60 min at 37°C (Xiang et al., 2014 (link)). The endometrial tissue underwent two rounds of digestion, and the dispersed cells were filtered through 40-μm sieves (BD Bioscience, San Jose, CA, United States). The red blood cells, cell debris, and cell clumps were removed by Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density-gradient centrifugation. Anti-CD45 antibody-coated Dynabeads (Invitrogen, Waltham, MA, United States) were used for the removal of leukocytes, and anti-CD326 antibody-coated microbeads (Miltenyi Biotec Inc., San Diego, CA, United States) were used to separate epithelial cells from stromal cell population. Freshly purified stromal cells were seeded onto 10-cm dishes (BD Biosciences, San Jose, CA, United States) coated with fibronectin (1 mg/ml, Invitrogen) containing growth medium (GM) with 1% penicillin (Invitrogen), 1% L-glutamine (Invitrogen), and 10% FBS (Invitrogen) in DMEM/F-12 (Sigma-Aldrich, St Louis, MA, United States). Stromal cells were cultured for 1–2 weeks in a humidified carbon dioxide incubator at 37°C. The medium was changed every 7 days.

The isolation procedure of endometrial cells was carried out as described [5 (link)]. The tissues were minced and digested with PBS containing collagenase type III (0.3 mg/ml, Worthington Biochemical Corporation, NJ, USA) and deoxyribonuclease type I (40 μg/ml, Worthington Biochemical Corporation) for 1 h at 37 °C. Red blood cells were removed using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density-gradient centrifugation. Leukocytes were excluded using anti-CD45 antibody-coated Dynabeads (Invitrogen, Waltham, MA, USA). Epithelial cells were separated from the stromal cells using anti-CD326 (EpCAM) antibody-coated microbeads (Miltenyi Biotec Inc., San Diego, CA, USA). Freshly isolated epithelial cells and stromal cells were used for coculture and collection of condition medium as describe below. Some of the spare purified stromal cells (6000–8000 cells/cm²) were plated into 100-mm petri dishes coated with fibronectin (1 mg/ml, Invitrogen) and cultured in growth medium containing 10% FBS (Invitrogen), 1% antibiotics (Invitrogen) and 2 mmol/L glutamine (Invitrogen) in DMEM/F12 (Sigma-Aldrich, St Louis, MA, USA) for 7–14 days in a humidified carbon dioxide incubator at 37 °C in 5% CO₂. The medium was changed every 7 days until the cells reached 90% confluence.