Cd83 apc

Cells were fixed with paraformaldehyde (PFA) and treated with either BSA (extracellular staining) or BSA/Saponin to measure intracellular staining. The following antibodies were used to detect Zika virus: anti-Flavivirus 4g2 mouse IgG2a (NovusBio), anti-Flavivirus 4g2 monoclonal rabbit (Absolute Antibody), anti-Zika virus monoclonal Rabbit (Genetex).
All other antibodies were anti-human: DC-SIGN mouse IgG1 (AZN-D1), anti-langerin mouse IgG1 (10E2) both in house made, PE conjugated CD207 (langerin), APC conjugated CD1a (BD Biosciences), CD86-FITC (BD Pharmingen), CD80-PE (BD Pharmingen), CD83-APC (BD Pharmingen), DC-SIGN-FITC (R&D systems), CD3-APC/Fire750 (Biolegend), CD11c-APC (Biolegend), PEcy7-HLA-DR (BD Pharmingen), APCcy7-CD14 (BD Biosciences), APCcy7-CD11c (Biolegend),APC-AXL (Thermofisher, PE-MerTK (Thermofisher) and anti-Tyro3 (Thermofisher). For secondary detection the following antibodies were used: AF488-conjugated goat anti-mouse IgG2a (Invitrogen), AF647-conjugated donkey anti-rabbit (Thermofisher), FITC-conjugated goat-anti-mouse IgM (Invitrogen), AF488-conjugated donkey anti-rabbit (Thermofisher).
Flow cytometric analyses were performed on a BD FACS Canto II (BD Biosciences) and data was analysed using FlowJo V10 software (TreeStar).

Immature and mature LCs were phenotyped using CD1a-APC (BD Pharmingen), langerin-PE (Novocastra), CD86-FITC (BD Pharmingen), CD80-PE (BD Pharmingen), CD83-APC (BD Pharmingen), CD4-AF488 (Biolegend), CD195 (CCR5)-PE (BD Pharmingen) and unlabeled CXCR4 (R&D systems) for which secondary detection with Goat-anti-Mouse-A488 (Invitrogen) was used. HIV-1 infection and transmission samples were stained for CD1a-APC (LC marker), CD3-PerCP (T cell marker), and p24-PE (HIV-1 envelope protein, Beckman Coulter). Immature vaginal LCs were further sorted with a FacsARIA 3 laser sorter (BD Biosciences) after staining for CD1a-APC into CD1a positive and CD1a negative fractions. Samples were analysed using FACSCanto II flow cytometers (BD Biosciences) and data analysis was carried out with FlowJo V10.

Flow cytometry was performed to assess the expression of DC- and T-cell surface markers as well as FluorDye signal intensity as described previously (34 (link)). For cell surface staining, directly labeled mAbs including CD14-FITC, CD25-V450, CD69-APC-Cy7, CD83-APC, CD86-PE, B7-H1-PE, CD3-PE, CD4-APC-H7, CD8-V500 (all from BD Biosciences, Heidelberg, Germany), and CD80-FITC (Beckman Coulter Inc, Indianapolis, USA) were used. In brief, cells were collected, washed with PBS and resuspended in FACS buffer (PBS supplemented with FCS, EDTA). The cell suspension was blocked for 10 min with human FCR blocking reagent (Miltenyi Biotech GmbH), incubated for 30 min with antibodies, washed 2× and re-suspended in FACS buffer. Afterwards, each sample was analyzed by a BD FACSCanto II flow cytometer and FlowJo software (both BD Biosciences). Results were expressed as medium fluorescence intensity (MFI) or percentage of cells.

Surface protein expression of CD11c, CD14, CD25, CD83, CD86 and HLA-DR of iDC, mDC and vitD3-tolDC was determined by flow cytometry. For each measurement, DC suspensions were incubated for 20 min, protected from light, with the adequate amounts of monoclonal antibodies anti-: CD11c PE-Cyanine dye 7 (PE-Cy7), CD14 Violet 450 (V450), CD25 allophycocyanin (APC), CD83 APC, CD86 fluorescein isothiocyanate (FITC) and HLA-DR Violet 500 (V500) (all of them from BD Biosciences). Afterwards, at least 10,000 CD11c⁺ events of each sample were acquired in a FACSCanto II flow cytometer and analyzed using FACSDiva software.

Differentiation and maturation of DCs exposed to Chlamydia or LPS and HIV-1 were analyzed by using anti-human CD11c-AlexaFluor488, HLA-DR-PerCP/Cy5.5, DC-SIGN-PE (Biolegend), CD86-BV421, CD83-APC, CD169-PE (BD Biosciences) on a FACS Verse flow cytometer (BD Biosciences). Cell surface expression of receptors for HIV and HIV-C binding was determined by flow cytometry as described using anti-human CD11b-APC, CD4 PerCP/Cy5.5 and DC-SIGN-PE (BioLegend). Data was analyzed using FACS DIVA software (BD Biosciences) and R.

To characterize monocyte subsets, freshly purified PBMCs were analyzed by six-color flow cytometry on FACS LSRII apparatus. The gating strategy was based on a previous report [15 (link)]. Monocytes were subdivided into three major subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and non-classical CD14⁺CD16⁺⁺ monocytes. The following anti-human mAbs were used: CD45-Amcyan (BD Biosciences), HLA-DR-PerCP (BD Biosciences), CD19-ECD (Beckman Coulter), CD14-QDot655 (Invitrogen), CD16-APC-H7 (Beckman Coulter, Villepinte, France). The Live/Dead blue Dye (Invitrogen) was used to exclude dead cells.
Samples of the purified monocytes used to generate MD-DCs and of the resulting MD-DCs were routinely stained with the following anti-human mAbs: CD14-FITC, CD11c-APC, CD40-PE, HLA-I-FITC, HLA-DR-PerCP, CD80-PE, CD83-APC and CD86-FITC (all from BD Bioscience) and analyzed by flow cytometry on FACS canto II apparatus (BD Biosciences).

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).