Pe conjugated anti il 10

Allophycocyanin-conjugated (APC) mAbs to Foxp3 and CD62L; Phycoerythrin-conjugated (PE) mAbs to CD4, CD44, TNF-α, and LAP; PerCPCy5.5-conjugated mAbs anti-CD4, IFN-γ, and rat IgG2a isotype control from BD Biosciences (San Jose, CA, United States) were used as markers for T cells. PerCP-Cy5.5-CD19, FITC-CD25, and PE-conjugated anti IL-10 from Biolegend (San Diego, CA, United States). For surface antigen detection, cells were labeled with monoclonal antibodies for 30 min at 4°C. For intracellular labeling of cytokines and transcription factors, a fixation/permeabilization kit (e-Bioscience, San Diego, CA, United States) was used after this step. Samples were then incubated for 30 min with a solution containing the appropriate antibodies. After washing with PBS containing 0.5% FBS, samples were fixed with 3% paraformaldehyde for 30 min, washed and stored in PBS at 4°C. Cells were acquired using a FACSCanto II cytometer (Becton Dickinson, East Rutherford, NJ, United States) and data was analyzed by FlowJo software (Tree Star, Ashland, OR, United States). At least 30,000 events were acquired for each analysis. Gating strategies are detailed in Supplementary Material (Supplementary Figure S1).

Spleens were collected and pressed through 70-μm cell strainers with complete medium (RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 1% 100 MEM non-essential amino acids, 10 mM HEPES, 55 mM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, all from Life Technologies, Grand Island, NY). Red blood cells (RBCs) were excluded using previously published methods (30 (link)). Single-cell suspensions were stimulated for 24 hours with 10 μg/ml LPS, followed by 5 hours of 50 ng/ml PMA and 500 ng/ml ionomycin, blocked for 10 minutes on ice with the Fc receptor block anti-CD16/32 (eBioscience), then stained for 15 minutes in the dark with fluorochrome-conjugated antibodies and analyzed with a BD FACS Aria II flow cytometer (BD Biosciences, San Jose, CA). Foxp3 Fixation/Permeabilization kit (eBioscience) was used for intracellular staining. To exclude dead cells, a Zombie Aqua fixable viability kit (Biolegend) was used. The following monoclonal anti-mouse antibodies were used in this study: AF700 or APC conjugated anti-CD19 diluted 1:800, PE-Cy7 conjugated anti-CD23 diluted 1:60, APC-Cy7 conjugated anti-CD21 diluted 1:80, FITC conjugated anti-CD24 diluted 1:200, PE conjugated anti-IL-10 diluted 1:80, BV711 conjugated anti-CD138 diluted 1:800, and APC conjugated anti-IgH (μ chain) diluted 1:800 (BioLegend).

The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: fluorescein isothiocyanate (FITC)-conjugated anti-HLA-DR; phycoerythrin (PE)-conjugated anti-CD86; and allophycocyanin (APC)-conjugated anti-CD83 (Miltenyi Biotec). APC-conjugated anti-CD3; and Alexa Fluor 488-conjugated anti-IFN-γ (BD Pharmigen). PE-conjugated anti-IL-10; Alexa Fluor 488-conjugated anti-IL-17A; peridinin-chlorophyll-protein (PerCP)-conjugated anti-CD4; Alexa Fluor 488-conjugated anti-FOXP3; PE-conjugated anti-CD127; and APC-conjugated anti-CD25 (Biolegend). Cells were washed with PBS/EDTA 2 mM/0.5% BSA and stained for 15 min at room temperature in the darkness. For analysis of FOXP3 expression in human T cells primed with hmoDCs, cells were first subjected to surface staining with anti-human CD127-PE, CD4-PerCP, and CD25-APC antibodies. After fixation and permeabilization, cells were stained with anti-human FOXP3-Alexa Fluor 488, according to manufacturer’s recommendations. For each staining, the corresponding isotype controls (IgG2A-FITC, IgG1-Alexa Fluor 488, IgG1-PE, IgG2A-PerCP, or IgG1-APC) were also assayed. Flow cytometry analysis was performed in a FACSCalibur in the Cytometry and Fluorescence Microscopy Unit at Complutense University of Madrid.