Pe conjugated anti cd86

The DC subsets were incubated overnight at 37°C with different stimuli in triplicate (CD1c⁺ mDCs, pDCs) or in duplicate (CD141⁺ mDCs). The next day, supernatants were taken and cells were labeled with PE-conjugated anti-MHC class I and FITC-conjugated anti-MHC class II (BD), PE-conjugated anti-CD80 (BD Biosciences), and PE-conjugated anti-CD86 (BD Biosciences). Marker expression was determined by flow cytometry (BD Calibur and FlowJo software). Supernatants were analyzed for IL-10 (eBioscience) and IL-12p70 (M122 and M121B by Pierce Endogen, standard by BD Biosciences) by standard sandwich ELISA. Depicted in Figure 2 is the cytokine production by 50,000 DCs in a volume of 100 µL. For CD141⁺ mDCs, in some instances fewer cells were cultured. In all instances, cytokine production per cell was calculated.

To study surface molecules expression, cells were washed with PBS, removed with a cell scraper, and labeled with human FITC-conjugated anti-CD14 (BioLegend, USA) or PE-conjugated anti-CD14 (BD Pharmingen, USA) and PE-conjugated anti-HLA-DR (BD Pharmingen, USA), PE-conjugated anti-CD86 (BD Pharmingen, USA) or FITC-conjugated anti-CD80 (BD Pharmingen, USA), and FITC-conjugated anti-CD40 (BD Pharmingen, USA) or PE-conjugated anti-CD69 (BD Pharmingen, USA) for 30 minutes at 4°C.
Cells were then washed twice with PBS + FCS 5% (200 ×g, 7 minutes) and resuspended in the same solution in the cold. Fluorescence measurements were performed using a FACScan or a FACSCalibur flow cytometer (Beckton, Dickinson and Company, USA). Fluorescence signals for no less than 10,000 cells were recorded in the monocyte gate, which was determined according to cell size and complexity parameters. Using these parameters, approximately 98% of the cells in this gate were CD14⁺. Data analyses were performed using Summit v4.3 software (Dako, USA).

Mononuclear cells from mouse uterine and placenta were prepared as previously described (Liu X. et al., 2014 (link)). Briefly, mice were sacrificed on Gd 14. The uterus and placenta were excised with surgical cuts. The pregnancy outcome was reflected by the total number of fetuses, fetal size, stillbirth, absorptive fetus, and hemorrhagic appearance. The uterus and placentas were washed with sterile PBS, minced into small pieces. Dispersed cells were collected by filtration through a 48 μm pore size stainless steel mesh. Thereafter, the mononuclear cells were obtained by density-gradient centrifugation and washed twice in cold PBS. The following fluorochrome-conjugated, mouse-specific mAbs were used in the assays: FITC-conjugated anti-CD11c (BD Biosciences, United States), Percp-cy5.5-conjugated anti-CD8a mAb (BD Biosciences, United States), PE-conjugated anti-CD80 (BD Biosciences, United States), PE-conjugated anti-CD86 (BD Biosciences, United States), PE-conjugated anti-I-Ad/I-Ed (MHC II) (BD Biosciences, United States), and PE-conjugated anti-LILRB4 (Biolegend, United States). Cells were incubated for 30 min at 4°C in the dark according to the manufacturer’s instructions. Subsequently, the cells were washed with cold PBS and analyzed using BD FACSAria flow cytometry and BD FACSDiva 7.0 software (Becton Dickinson, United States).

Astragalin was modified into astragalin-galactoside (Ast-Gal) using β-galactosidase from Bacillus circulans, and purified by the medium pressure chromatography with silica C-18 column followed by Sephadex LH-20 column [25 (link)]. Ast-Gal was identified by nuclear magnetic resonance to be kaempferol-3-O-β-d-glucopyranosyl-(1->6)-β-d-galactopyranosyl-(1->4)-β-d-galactopyranoside. The water solubility of Ast and Ast-Gal were 28.2 ± 1.2 mg/L and 38,800 ± 2.8 mg/L, respectively. Anti-CD3ε, anti-CD28, and anti-IL-12p70 antibodies were purchased from BD Biosciences (San Diego, CA, USA). Mouse recombinant IFN-γ and IL-4 were purchased from PROSPEC (East Brunswick, NJ, USA). Mouse recombinant IL-6 and TGF-β were purchased from PEPROTECH (Rocky Hill, NJ, USA). FTIC-conjugated anti-CD4, FTIC-conjugated anti-MHC Class II, FTIC-conjugated anti-IgG 2b/k, PE-conjugated anti-IFN-γ, PE-conjugated anti-IL-4, PE-conjugated anti-CD80, PE-conjugated anti-CD86, PerCP-Cy5.5-conjugated anti-CD4, and APC-conjugated anti-IFN-γ antibodies were purchased from BD Biosciences. PE-conjugated anti-Rat IgG2a, PE-conjugated anti-IL-13, APC-conjugated anti-IL-17A, and APC-conjugated anti-CD11c were purchased from eBioscience (San Diego, CA, USA).