Isolation of Immune Cells from Porcine Maternal-Fetal Interface

The procedure for the isolation of immune cells from the porcine maternal-fetal interface has been described previously (37 (link)). In brief, ME and FP tissues were cut into small pieces and incubated in tissue digestion medium [RPMI-1640 supplemented with 2% (v/v) heat-inactivated fetal calf serum (FCS; Sigma-Aldrich, Schnelldorf, Germany), 25 U/mL DNase type I (Thermo Fischer Scientific), 300 U/mL Collagenase type I (Thermo Fisher Scientific), 100 IU/mL penicillin (PAN-Biotech), and 0.1 mg/mL streptomycin (PAN-Biotech)] for 1 h at 37 °C and constant mixing. Remaining larger pieces of tissue and dead cells were removed by draining the cell suspensions through a coarse-meshed sieve and subsequent filtering through a layer of cotton wool. Suspensions were centrifuged (350 × g, 10 minutes, 4°C), resuspended in 40% Percoll (13 mL, Thermo Fisher Scientific), underlaid with 70% Percoll (13 mL, Thermo Fisher Scientific), and subjected to density gradient centrifugation (920 × g, 30 minutes, room temperature). Isolated leukocytes were washed four times (phosphate-buffered saline (PBS, 2x), RPMI-1640 + 5% FCS (1x), and RPMI-1640 + 10% FCS (1x)) and immediately used for immune phenotyping.