Ls004182

Adult microglia were isolated using magnetic-activated cell sorting (MACS) as described before⁷⁰ . Briefly, anesthetized mice were thoroughly transcardially perfused with cold PBS to remove circulating blood cells in the CNS. Dissected brains were chilled on ice and minced in digestion media containing 0.2% collagenase type 3 (LS004182, Worthington) and 3 U/mL dispase (LS02104, Worthington). After 37 °C incubation for 45 min, digestion was inactivated by 2.5 mM EDTA (15575020, Thermofisher) and 1% fetal bovine serum (10082147, Thermofisher). Digested brain tissues were triturated by serological pipette to the cell suspension and passed through a 70 μm cell strainer. Myelin in the cell suspension was depleted by myelin removal beads (130-096-733, Miltenyi Biotec) and magnetic LD columns (130-042-901, Miltenyi Biotec). Adult microglia were finally enriched from the eluant by CD11b MicroBeads (130-049-601, Miltenyi Biotec) and magnetic MS column (130-042-201, Miltenyi Biotec) for RNA isolation.

The use of human tissue or cells was approved by the ethics committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. Written informed consents were obtained from women aged 25–40 years old who underwent hysteroscopic examination of endometrium with biopsy 3–7 days after the completion of menstruation. Endometrial samples (about 0.3 g) were collected from those having regular menstrual cycles and no evidence of neither endometritis nor endometriosis. The tissue was rinsed with sterile PBS for 3 times, then cut into <1-mm³ pieces before dispersion in DMEM/F12 medium supplemented with collagenase III (0.5 mg/ml, Worthington LS004182) and DNase (0.2 mg/ml, Worthington LS002139) at 37°C in a shaking water bath for 60 min. Enzyme dispersion was terminated by adding an equal volume of DMEM/F12 with 10% FBS. The isolated endometrial stromal cells were obtained by using a 70-µm sieve to filter the undigested tissue pieces. The effluent was centrifuged at a speed of 800 rpm for 5 min. The cells were seeded onto a 10-cm dish. After 24 h, the culture medium (DMEM/F12 with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin) was changed and the adherent human endometrial stromal cells (HESCs) were further cultured till 90–100% confluence for passages. Cells from Passage 4 (P4)–P7 were used for experimentations.