H3 thymidine

MDSCs were isolated from the peritoneal cavities of BALB/c mice at 12 h after the transplantation of endometrial implants, and G-MDSCs and M-MDSCs were sorted from the peritoneal MDSC samples by MoFloAstrios EQ Cell Sorter (Beckman Coulter, Brea, CA, USA) (Supplementary Fig.9). The purity of sorted MDSC subsets was greater than 95%. MDSCs, G-MDSCs and M-MDSCs were cultured in 96-well round bottom plates in the presence of different ratios with 5×10⁵ purified T cells stimulated with 4 mg/mL Con A (Sigma-Aldrich, St. Louis, MO, USA). The cells were cultured in 200 μL of RPMI-1640 medium supplemented with 10% FBS, 10mM HEPES, 1mM penicillin–streptomycin and 50 mM β-mercaptoethanol (Invitrogen, Carlsbad, CA, USA) at 37°C in a 95% humidified incubator with 5% CO₂. After culture for 72 h, 10 μCi of [H3]-thymidine (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. After culture for additional 12 h, the cells were collected by cell harvester and the incorporation of [H3]-thymidine was detected in MicroBeta² Plate Counter (Perkin Elmer, Waltham, MA, USA).

Bone marrow-derived dendritic cells (BMDCs) were isolated from tibias and femurs of 8-week-old female C57/BL6 mice.^{37 (link)} Cells were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF; Invitrogen, CA USA) (10 ng/mL) for 6 days, to induce differentiation of DCs.^{37 (link)} For the proliferation assay, freshly isolated CD4⁺ T cells from lymph node cells of OT-II transgenic mice (kindly provided by Dr C. Lowell, University of California, USA) were co-cultured with LPS (100 ng/mL)-stimulated DCs and 1 μg/mL with OVA323-339 peptide (Genomics, Taiwan) at 1:4 DCs/T cell ratio by measuring uptake of [³H]-thymidine (Amersham Pharmacia Biotech, New Jersey, USA) in TopCount (PerkinElmer Life Sciences, Palo Alto, CA, USA).^{38 (link)} T cell proliferation of splenocytes was measured by [H³]-thymidine as described above.