Phorbol 12 myrstate 13 acetate pma

Uteri from pregnant mice were dissected free from the mesometrium and the fetal and placental tissues were carefully removed from the uterus. Washed and Minced uteri were digested in RPMI 1640 (HyClone, U.S.A.) supplemented with collagenase type IV (1.0 mg/ml, Worthington Biomedical, U.S.A.) and DNase I (150 U/ml, Applichem, Germany) for 30 min at 37 °C with gentle agitation. Cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml amphotericin B at 37 °C in 5% CO₂ for 2 h to remove adherent stromal cells. Then the cell suspensions were collected and Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, BioLegend, U.S.A.), ionomycin (1 μg/ml, Biolegend, U.S.A.) and brefeldin A (10 mg/ml, BioLegend, U.S.A.) were added in the culture for 4 h for intracellular cytokine analysis.

Freshly isolated trophoblasts were seeded at a density of 2 × 10⁵ cells/ml per well in Matrigel (Coring, USA)-coated 24-well plates overnight. The cells were then washed twice with phosphate-buffered saline (PBS, HyClone, USA). Equal numbers of dCD8⁺ T cells or pCD8⁺ T cells were added to each well. In some wells, anti-HLA-C (10 μg/ml, clone W6/32; Biolegend, U.S.A), HLA-G (10 μg/ml, clone 87G; Biolegend, USA) were added. dCD8⁺T cells were also cultured with plate-bound anti-CD3 antibody (OKT-3; 5 μg/ml, Biolegend, USA) plus soluble anti-CD28 antibody (28.2; 1 μg/ml, Biolegend, USA), or HTR8/Svneo cells or DSCs for 48 h. In some wells, dCD8⁺ T cells (2 × 10⁵ cells) were plated in the upper chamber (0.4 mm pore size cell culture inserts, Millipore, Germany), while trophoblasts were plated in the lower chamber to establish indirect cell contact. Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, Biolegend, USA), ionomycin (1 μg/ml, Biolegend, USA) and brefeldin A (10 mg/ml, BioLegend, USA), were added 4 h before the end of the 48 h culture for intracellular cytokine analysis. The cells were then harvested for flow cytometry analysis.