Isolation and Analysis of Murine Endometrial Stromal Cells

Procedures were principally performed as described [39 (link)]. Briefly, murine uteri were minced into small pieces and incubated in PBS containing 1 mg/mL collagenase type IV and 40ug/ml DNase I for 45 min at 37 °C with shaking at 100 rpm. For flow cytometric analyses, cell suspensions were incubated with anti-CD16/32 for Fc blocking (101319, BioLegend), and stained with PE/cyanine7-conjugated anti-CD45 (103113, BioLegend), PerCP/Cyanine5.5-conjugated anti-CD11b (101227, BioLegend), and PE-conjugated anti-Ly6G (127607, BioLegend) mAbs for 30 min on ice. For eutopic stromal cell and ectopic stromal cell isolation, cell suspensions were strained by using 70 μm nylon cell strainers. The generated cell suspensions were strained by using 70 μm and 40 μm nylon cell strainers and twice washed by centrifugation at 300 g. The final pellet containing the endometrial stromal cells was cultured at 37 °C with 5% CO₂ in the DMEM/F-12 GlutaMAX (SH30023.01, Hyclone) medium supplemented with 10% fetal bovine serum (FBS) and the antibiotic/antimycotic mix. The cells were allowed to adhere for 24 h, and non-adherent cells were removed by replacing the medium. Upon reaching 70–90% confluence, adherent cells were harvested by trypsinization and subcultured at a 1:3 ratio. To measure glucose uptake, eutopic stromal cells and ectopic stromal cells were incubated with 100 mM 2-NBDG (N13195; Invitrogen) for 10 min at 37 ℃. Samples were acquired by a BD FACSVerse flow cytometry, and data were analyzed with FlowJo software (TreeStar).