Macsmix

Live CD4⁺ human tonsillar lymphocytes were stained with the anti-human IL-10 catch reagent (Miltenyi) at 9 million cells per 100 µl. After 10 min on ice, the cells were diluted 1:20 in RPMI supplemented as above. Cells were stimulated with PMA (100 ng/ml) and ionomycin (500 ng/ml) and incubated for 2 h in a 37°C incubator with 5% CO₂, rotating slowly using a MacsMix (Miltenyi). Cells were then stained as above using the anti-human IL-10 detection antibody (Miltenyi).

The cytokine secretion assay on pentamer⁺ CD8⁺ T cells was performed as described previously (29 (link)). Briefly, PBMCs were coupled with capture reagents (Cytokine Secretion Assay kit, Miltenyi Biotec, Auburn, AL, USA) for human IFN-γ and TNF-α under stimulation from the peptide mixture. The cells were incubated in a closed tube for 45 min at 37 degrees Celsius under slow continuous rotation using the MACS mix (Miltenyi Biotec). After a washing step, cells were resuspended in cold medium containing IFN-γ and TNF-α detection antibodies. Subsequently, the cells were resuspended in MACS buffer containing antibodies specific for surface markers, including pentamer, and flow cytometry was performed on a BD LSR II cytometer.

Endometrial tissues were minced with scissors and digested using 5% collagenase type II and 40 µg/ml deoxyribonuclease type I (DNAse I) (Worthington-Biochemical Corporation) at 37 °C in Dulbecco’s modified Eagle’s Medium/F12 medium (DMEM/F12) containing 15 µM Hepes buffer (Invitrogen) in a humidified incubator at 37 °C on a rotating MACSmix (Miltenyi Biotech) for 60–90 minutes. Dissociated stromal cells were separated from collagenase resistant epithelial cells (as clumps) using a 40 µm sieve (BD Bioscience-Durham) and red blood cells removed by density gradient centrifugation using Ficoll-Paque (GE Healthcare Bioscience-Bio-Sciences AB) as previously described^{44 (link)}. Single cell suspensions of stromal cells were incubated with PE-conjugated SUSD2 (formerly W5C5) antibody (2 µg/ml) (Biolegend) for 30 minutes at 4 °C followed by incubation with anti-PE labelled magnetic beads (Miltenyi Biotec) for 20 minutes and SUSD2⁺ eMSC were selected using a column and magnet (Miltenyi Biotec). SUSD2⁺ eMSCs were cultured in DMEM/F12 medium containing 10% Fetal Calf Serum (FCS) (Invitrogen), 1% antibiotic-antimycotic (Life Technologies) and 2 mM glutamine (Life Technologies) for 2–4 passages. We have previously shown that this protocol isolates eMSC which robustly express the typical MSC markers when cultured (ie CD29, CD44, CD73, CD90, CD105 > 90%; CD31, CD34, CD45 < 5%).

We resuspended the washed platelets in M199 (ThermoFisher Scientific, Waltham, MA, USA) cell culture medium without added substances. We used a rotator designed to be used in a cell culture incubator (MacsMix, Miltenyi Biotec, Bergisch Gladbach, Germany) to prevent platelet sedimentation during the suspension culture. Tubes containing the platelet suspension were placed properly in the adaptor of the rotator and set to gentle rotation. The rotator and the culture tubes were then put into a cell culture incubator for six hours. Before starting a culture, an aliquot of platelet suspension in liquid medium was collected, and the platelet count was measured in triplicate using the automated hematology analyzer (Coulter counter, Advia 2120i, Siemens). After six hours of culture, platelet counts were measured in triplicate and compared to the initial count.