G molecules in dM or uM, cells were permeabilized (Cytofix/Cytoperm
G molecules in dM or uM, cells were permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and incubated with Alexa Fluor 647-conjugated Akt (pS473) antibody (561670) and Alexa Fluor 488-conjugated Stat6 (pY641) antibody (612600 for dM; 558243 for uM) (Phosflow antibodies had been from BD Biosciences) . Isolation and culture of human trophoblasts, DSCs and DLCs. The villi and decidua tissues in the first-trimester pregnancy had been put immediately into ice-cold Dulbecco’s modified Eagle’s medium (DMEM high D-glucose; Gibco, Grand Island, NY, USA), transported to the laboratory within 30 min just after surgery Cell Death and DiseaseRANKL regulation of decidual M Y-H Meng et aland washed in Hank’s balanced salt option for isolation of human trophoblasts from villi, and DSCs and DLCs from deciduas according to a previously described approach.46 This process supplies a 95 purity of vimentin-cytokeratin (CK)7+ trophoblast cells and higher than 98 vimentin+CK7- DSCs and CD45+ DLCs, which was confirmed by FCM analysis. Enzyme-linked immunosorbent assay (ELISA). Cytokine concentrations were measured using ELISA kits (R D Systems). Detection of RANK expression on peripheral blood mononuclear cells (PBMCs) and DLCs. The PBMCs have been isolated from the peripheral blood of pregnant girls who had been terminated for nonmedical causes. Next, FCM was CDCP1, Mouse (Biotinylated, HEK293, His-Avi) performed to analyze the expression of RANK on pMo and dM by labeling anti-human CD14, RANK and CD45 antibodies. Also, we further evaluated the phenotype of RANK+ and RANK- pMos and dM purified from PBMC and DLC (n = 24) by FCM. Purification of dM and decidual naive T cells. We isolated dM and decidual naive T cells from DLCs by MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and performed FCM analysis with typical protocols. RNase Inhibitor web RANKL-overexpressed JEG-3 cells and DSCs. We obtained RANKLoverexpressed JEG-3 cells and DSCs by transfection with pcDNA(+)-RANKL plasmid, and also the outcomes were confirmed by FCM analysis. The pcDNA(+)-RANKL plasmid and pcDNA(+)-vector plasmid have been from GeneChem Co., Ltd (Shanghai, China). Co-culture of trophoblasts/JEG-3 cells, DSCs and dM. The dM have been cultured in culture medium, directly contacted with major trophoblasts/JEG-3 cells and or DSCs at a 1 : 1 : 1 ratio. Moreover, 5 g/ml anti-human RANKL neutralizing antibody (AB626, R D Systems), one hundred ng/ml rhOPG protein (185OS-025, R D Systems), 10 M LY294002 (Cells Signal Technologies, Danvers, MA, USA) or 21 nM STAT6 signal inhibitor (STAT6i) AS1517499 (Axon Medchem, Groningen, The Netherlands) was added to the co-culture unit. Right after 48 h, the expression of RANKL on trophoblasts and DSCs, the expression of RANK, and the M1 phenotype and M2 phenotype on dM were analyzed by FCM, plus the concentration of IL-10, IL-12p40 and IL-23 in the supernatants was detected by ELISA (R D Systems). The intracellular phosphorylation level of Akt and STAT6. The intracellular phosphorylation amount of Akt and STAT6 in dM right after 24-h culture with JEG-3 and DSCs was analyzed employing BD Phosflow antibodies, in line with common protocols. The transcription of Jmjd3, IRF4 and IRF5. Just after 24-h co-culture, the transcription level of Jmjd3, IRF4 and IRF5 in dM was analyzed by real-time PCR, according to common protocols. The primer sequences had been made and synthesized by TaKaRa Biotechnology Co., Ltd (Tokyo, Japan) as described in Supplementary Table 1. Co-culture of dM and decidual naive T cells. Following 48 h of culture with trophoblasts/JEG-3 cells and DSCs, CD.
