Multicolor flow cytometry was carried out on fresh Ficoll-prepared (TBD Sciences, Tianjing, China) PBMCs. After suspension in PBS, PBMCs (100 μL) were separately added to two tubes (1 × 106–2 × 106 PBMCs per sample). The first tube was incubated with FITC-Lineage (CD2, CD3, CD14, CD16, CD19, CD235a, and CD56), APC-HLA-DR, PE-Cy7-CD11c, and Percp-Cy5.5-CD123 mAbs (all eBioscience, San Diego, CA, USA). The PBMCs defined as Lineage−HLA-DR+CD123brightCD11c− were pDCs. The other tube was used to identify CD4+CD25+Foxp3+ Tregs and analyze their ICOS expression. Briefly, after CD4-FITC (BD Pharmingen, San Diego, CA, USA), CD25-PE-Cy5 (BD Pharmingen) and ICOS-PE-Cy7 (eBioscience) surface staining, PBMCs were washed and fixed with fixation/permeabilization solution (eBioscience), then incubated with anti-Foxp3-PE mAbs (eBioscience). After washing, stained cells were analyzed on a flow cytometer (LSR-II; BD Biosciences) using BD FACSDiva Software. Lymphocytes (1–3 × 104) were gated to analyze the Tregs. Considering the small proportion of pDCs, 100 000–200 000 events in the lymphocytes and monocytes were accumulated as the acquisition gates. The appropriate isotype was used.
Cd25 pe cy5
Manufactured by BD
Sourced in United States, Germany
The CD25-PE-Cy5 is a fluorescent-conjugated antibody used for the detection and analysis of CD25 (Interleukin-2 Receptor Alpha Chain) expression on cells. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 fitc, by BD (2 mentions)
Cd83 apc, by BD (2 mentions)
Facsdiva software, by BD (1 mentions)
Cd11c pe cy7, by Thermo Fisher Scientific (1 mentions)
Hla dr apc, by Thermo Fisher Scientific (1 mentions)
Anti foxp3 pe mab, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization solution, by Thermo Fisher Scientific (1 mentions)
Icos pe cy7, by Thermo Fisher Scientific (1 mentions)
Fitc lineage, by Thermo Fisher Scientific (1 mentions)
Cd14 fitc, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd86 pe, by BD (1 mentions)
Cd19 bv510, by BioLegend (1 mentions)
Cd27 pe cy7, by BioLegend (1 mentions)
Igm fitc, by Thermo Fisher Scientific (1 mentions)
Cd40 pe, by BD (1 mentions)
7 aad staining, by BD (1 mentions)
Cd5 fitc, by Thermo Fisher Scientific (1 mentions)
Cd24 apc, by Thermo Fisher Scientific (1 mentions)
6 protocols using cd25 pe cy5
1
Multicolor Flow Cytometry for pDCs and Tregs
2
Phenotyping Monocyte Surface Markers
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Monocytes were stained with fluorochrome-conjugated antibodies CD14-FITC, CD25-PE-Cy5, CD83-APC (BD) and CD80-PEVio770 and CD86-PE (Miltenyi Biotech, Bergisch Gladbach, Germany) for 15 min at room temperature, washed with PBS and analysed in a Canto II flow cytometer (BD).
3
Phenotyping of B cell subsets
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For phenotyping of B cells, CD9+ and CD9− B cells, the antibodies used were as follows: CD27-PE-Cy7 (clone 0323, Biolegend), CD38-PE (clone HB7, Biolegend), CD19-BV510 (clone HIB19, Biolegend), CD24-APC (clone SN3 A5-2H1D, eBioscience, San Diego, CA, USA), HLA-DR-PERCP (Clone L243, Biolegend), CD40-PE (Clone 5C3, BD Biosciences), CD86-PE (Clone 2331, BD Biosciences), CD25-PE/Cy5 (Clone M-A251, BD Biosciences), CD69-APC (Clone FN50, Biolegend), IgD-APC-Cy7 (Clone, IA6-2, Biolegend), IgM-FITC (Clone, SA-DA4, eBioscience), CD5-FITC (Clone, L17F12, eBioscience), CD10-APC (Clone, LT10, ImmunoTools, Friesoythe, Germany), CD20-PE-Dy647 (Clone, LT20, ImmunoTools) and CD21-FITC (Clone, LT21, ImmunoTools). Viability was assessed by 7AAD staining (BD Biosciences). All flow cytometric acquisition was performed with FACS Canto II, BD Biosciences. The data analysis was performed using FlowJo V7 (TreeStar Inc., Ashland, OR, USA). The gating strategy for the Bregs and single markers can be found in Supplementary Figure S1 .
4
Characterizing Immune Cell Subsets in PBMCs
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The peripheral blood mononuclear cells (PBMCs) were separated from freshly collected samples and were stained for the analysis of thymic output, immune activation, and regulatory T cell (Treg) subsets by flow cytometry (Zhou et al., 2013) . The antibodies (CD4-FITC, CD4-PE, CD8-PerCp-Cy5.5, CD8-FITC, CD45RA-APC, CD45RA-FITC, CD31 PE, CD25 PE-Cy5, HLA-DR Percp, and CD38 APC) were purchased from BD Biosciences. Data acquisitionwas performed on a FACS-Calibur flow cytometer (BD Biosciences) and data were analyzed using FlowJo Software (Tree Star, Ashland, OR, USA).
Real-time PCR for the detection of interferon-stimulated genes (ISGs)
Total RNA was extracted from PBMCs and real-time PCR was performed as described previously (Fernandez et al., 2011) . All primers used for detection are listed in Supplementary material Table S1.
Real-time PCR for the detection of interferon-stimulated genes (ISGs)
Total RNA was extracted from PBMCs and real-time PCR was performed as described previously (Fernandez et al., 2011) . All primers used for detection are listed in Supplementary material Table S1.
5
Flow Cytometry of Memory T Cell Subsets
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Samples of fresh ACD-anticoagulated whole blood were stained with monoclonal antibodies, lysed and fixed, as above. Monoclonal antibodies used to study subsets of CD4 memory T cells were: CD3-PerCP-Cy5.5, CXCR3-Alexa Fluor 488 and -PE-CF594, integrin ß7-APC, CD4-Alexa Fluor 700, CD45RO-FITC, CD45RA-PE-CF594; CD49d-PE, CD25-PE-Cy5, CD8-BV786 (BD Biosciences), CD45RO-ECD (Beckman Coulter); CD49d-BV510, integrin ß7-biotin, CCR6-BV421, CD127-PE-Cy7 (BioLegend); CD161-PE, -APC or PE-Vio-770 (Miltenyi Biotec, Bergisch Gladbach, Germany); and CD62 L-APC-Cy7 (eBiosciences). A total of 400,000 events were collected, and a manual forward and side scatter gate was drawn to include lymphocytes, then gated sequentially on CD31, on CD41, and then on CD45RO1 cells using FlowJo, then exported as an FCS file using FlowJo.
6
Immature Dendritic Cells Capture Microvesicles
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To assess the ability of iDCs to capture MVs, 10 5 iDCs were incubated at 37 • C in 5% CO 2 with PKH-67 labelled MVs (25 g or 12.5 g) at a final volume of 150 l complete medium. As a control, iDCs were incubated at 4 • C. Several incubation times were assessed in the different experiments.
After incubation, cells were extensively washed in cold PBS. At this point, they were either stained for capture and phenotype analysis by flow cytometry or left in complete medium at 37 • C for a further 24 h. After culture, cells were assessed for expression of both activation markers and for allostimulation capabilities. For phenotype analysis, the following murine mAbs were used (BD Biosciences) CD83-APC, HLA-DR-APC-H7, CD25-PE and CD25-PE-Cy5. Isotype-matched mAbs were used as controls. All analysis was performed in a FACS Canto II flow cytometer (BD Biosciences) and analysed using FlowJo software. For inhibition experiments, cells were treated for 30 min at 37 • C with cytochalasin D (Calbiochem, Germany) at the indicated concentrations prior to the addition of MVs.
After incubation, cells were extensively washed in cold PBS. At this point, they were either stained for capture and phenotype analysis by flow cytometry or left in complete medium at 37 • C for a further 24 h. After culture, cells were assessed for expression of both activation markers and for allostimulation capabilities. For phenotype analysis, the following murine mAbs were used (BD Biosciences) CD83-APC, HLA-DR-APC-H7, CD25-PE and CD25-PE-Cy5. Isotype-matched mAbs were used as controls. All analysis was performed in a FACS Canto II flow cytometer (BD Biosciences) and analysed using FlowJo software. For inhibition experiments, cells were treated for 30 min at 37 • C with cytochalasin D (Calbiochem, Germany) at the indicated concentrations prior to the addition of MVs.
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