Collagenase type a1

Xenopus laevis oocytes and RNAs were prepared as previously described in detail (Bossi et al. 2007a). Oocytes were obtained from mature female of X. laevis; the frogs were anesthetized in MS222 (tricaine methanesulfonate salt; Sigma, Milan, Italy, www.sigmaaldrich.com) 0.10% w/v solution in tap water and portions of the ovary were removed through an incision on the abdomen. The oocytes were treated with collagenase Type A1 (1 mg/mL; Sigma) in calcium‐free solution ND96 for at least 1 h at 16°C. Healthy oocytes were selected and maintained at 16°C in MBS medium (modified Barth's saline solution). After 24 h, oocytes were injected with 50 nL of the in vitro synthesized cRNA (12.5 ng/oocyte) using manual Drummond injection system (Drummond Scientific Company, Broomall, PA, www.drummondsci.com). The oocytes were incubated at 16°C for 4–6 days in MBS before electrophysiological studies.

Endometrial tissue was obtained at day 15 of the menstrual cycle from healthy donors aged 18–35 years-old. Subjects diagnosed with endometriosis and/or endometritis were excluded. The study was carried out in accordance with the recommendations of local Ethical Committee at IVI Valencia, Spain with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the local Ethical Committee at IVI Valencia, Spain (study code: 1404-FIVI-015-CS).
Endometrial samples were mechanically disaggregated, digested with Collagenase type A1 (Sigma) and subjected to gravity sedimentation to separate the epithelial and stromal fractions as previously described (Simón et al., 1997 (link)). The epithelial fraction was plated in 24-well plates and cultured in hEEC medium (75% DMEM, 25% MCDB-105, 10% FBS, 5 pg/mL insulin, and 0.1% fungizone and gentamicin) until they reached a confluence of 80–90%. Then, the cells were washed twice with DMEM basal media (Sigma-Aldrich) to deplete from residual antibiotic and cultured in antibiotic-free media for an additional 8 h before colonization assays.