Dulbecco modified eagle medium (dmem)

Confluent MSCs, at passages 3–5, were washed in DMEM and cultured for 24 h in minimal base medium consisting of DMEM (Stem Cell Technologies, London, UK) supplemented with 1% insulin-free Sato (containing 100 μg/ml bovine serum albumin, 100 μg/ml transferrin, 0.06 μg/ml progesterone, 16 μg/ml putrescine, 0.04 μg/ml selenite, 0.04 μg/ml thyroxine, 0.04 μg/ml tri-iodothryonine), 1% holo-transferrin, 1% penicillin/streptomycin and 0.5% L-glutamine (Sigma-Aldrich, Gillingham, UK). The medium was then removed prior to being used within culture experiments. MSC conditioned medium in this study was prepared from >10 independent MSC samples.

Mammary tumors developed in PyMT/+ and CIRP/PyMT mice were isolated from the mice at necropsy (14 weeks of age). The tumors were washed in PBS, cut into 6–8 roughly equal pieces, and placed in a 10 mL digestion solution of DMEM/F12 + 5% fetal bovine serum with 1× collagenase/hyaluronidase in DMEM (Stemcell Technologies Cambridge, MA, USA), 0.05 U/mL Dispase (Stemcell Technologies Cambridge, MA, USA), and DNase I Solution (Stemcell Technologies Cambridge, MA, USA). The tumors were incubated at 37 °C for 100 min and briefly vortexed at 60% power every 10 min. The resulting cell suspension was then vortexed at full power for 30–45 s and then strained through a 70 μm cell strainer. The cells were then pelleted and washed in PBS twice. The cells were then resuspended in flow cytometry staining buffer containing PBS with 2 mM EDTA and 5% FBS.

Whole implantation sites on GD 9.5 were collected in sterile saline and placed under a dissection microscope. After removing the nascent embryo and placenta, decidual tissue was isolated, minced, and then digested in Gentle Collagenase/Hyaluronidase in DMEM (07919, StemCell Technologies) for 2 h at 37°C with moderate agitation as per the manufacturer’s instructions. Digested tissue was passed through a 100 μm cell strainer, and the resulting single cell suspension was layered over Lymphoprep (StemCell Technologies). After centrifugation, the buffy coat containing platelets, leukocytes, and other mononuclear cells was carefully aspirated, washed, and resuspended in RPMI-1640 medium supplemented with 1% fetal bovine serum (FBS) and 10 ng/mL rat recombinant IL-15 (PeproTech). Cells were then placed in cell culture plates at 1.0 × 10⁶ cells/mL in a gas and temperature-controlled incubator (5% CO₂, 37°C) for 24 h. After the incubation, conditioned media containing suspended cells were removed and centrifuged at 300 × g for 10 min. The supernatant was aliquoted and stored at −20°C as a source of decidual conditioned media and to measure levels of OPN. RNA was isolated from both the pelleted cells and the cells that adhered to the cell culture plate to evaluate expression of NK cell markers and Spp1.