Isolation of Uterine Luminal Epithelial Cells

Uterine luminal epithelial cells were isolated as previously described (Nallasamy et al., 2012 (link)). The uteri from the estrous mice or PD4 were cut longitudinally, washed in HBSS, incubated in 0.2% (W/V) trypsin (0458, Amresco, Cleveland, USA) and 6 mg/ml dispase (Roche Applied Science,4942078001, Basel, Switzerland) in 4.3 ml HBSS for 1.5 hr at 4℃, 30 min at room temperature, and 10 min at 37℃. After rinsing in HBSS, the epithelial cells were precipitated in 5% BSA in HBSS for 7 min. After the collected epithelial cells were cultured in DMEM/F12 (D2906, Sigma-Aldrich) with 10% heat-inactivated fetal bovine serum (FBS) in a culture plate for 30 min, the unattached epithelial cells were transferred into new culture plates precoated with ECM (1:100, E0282, Sigma-Aldrich) and cultured at 37 °C for 1 hr. Luminal epithelial cells were treated with TNF (10 and 100 ng/ml, 410-MT-010, R＆D systems) in DMEM/F12 with 2% charcoal-treated FBS (cFBS, Biological Industries, Cromwell, CT).