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5 protocols using cd45ra pe vio770

1

Multicolor Flow Cytometry Analysis of Lymphocyte Subsets

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Multicolour flow cytometric analysis was performed using the following fluorochrome-labelled anti-human monoclonal antibodies for measuring lymphocyte Th and Tfh subsets (CD4-APC-Vio770, CD45RA-PE-Vio770, CCR6-APC, CXCR3-VioBright FITC CXCR5 PerCP-Cy5.5, CCR4-PE) all from Miltenyi biotech. Tregs and Tfr were detected using BD Human Regulatory T Cell Cocktail (BD, San Jose, CA, USA), including CD4-FITC (clone SK3), CD25-PECy7 (clone 2A3), CD127 Alexa Fluor® 647 and adding CXCR5 PerCP-Cy5 and CD8 horizon450 (the last two from Biolegend). Bregs were detected using the following fluorochrome-labelled anti-human monoclonal antibodies: CD38-FITC CD1d-PE, CD19-PE-Cy7, CD5-PerCP-CY5.5, CD24-APC-H7 and CD27- BV510, all from BD Biosciences (San Jose, CA, USA).
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2

Comprehensive Immunophenotyping of Activated T Cells

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CD3-FITC (Clone: REA613, Miltenyi Biotec, catalog no. 130-113-138);
CD4-PerCp-Vio700 (Clone: REA623, Miltenyi Biotec, catalog no. 130-113-228);
CD69-VioGreen (Clone: REA824, Miltenyi Biotec, catalog no. 130-112-611);
CD154-APC (Clone: REA238, Miltenyi Biotec, catalog no. 130-113-610);
CD154-PE (Clone: 5C8, Miltenyi Biotec, catalog no. 130-092-289); CD137-PE
(Clone: 4B4-1, Miltenyi Biotec, catalog no. 130-119-885); CD25-PE-Vio615
(Clone: REA945, Miltenyi Biotec, catalog no. 130-115-537); CD45RA-PE-Vio770
(Clone: REA1047, Miltenyi Biotec, catalog no. 130-117-746); CCR7-VioBlue
(Clone: REA546, Miltenyi Biotec, catalog no. 130-117-353); interferon-gamma
APC (Clone: 4S.B3, BD Biosciences, catalog no. 551385); Foxp3-Alexa Fluor
647 (Clone: 236A/E7, BD Pharmingen, catalog no. 561184); LIVE/DEAD fixable
near-IR dead cell stain kit (ThermoFisher Scientific, catalog no. L10119);
FcR Blocking Reagent (Miltenyi Biotec, catalog no. 130-059-901).
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3

Multicolor Flow Cytometry for Immune Cell Profiling

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Multicolor flow cytometric analysis was performed, and the gating strategy is reported in the Supplementary Materials. The first tube was to detect B cell subsets and included the following mAbs: CD19-PE-Cy7 (SJ25C1), CD24-APC-Cy7 (ML5), CD38-FITC (HB-7), CD5-PerCP-Cy5.5 (L17F12), CD1d-PE (all from BD Biosciences, San Jose, CA, USA), CD45-APC (REA747 Miltenyi Biotec S.r.l., 40133 Bologna, Italy), and CD27-BV510 (M-T271 BioLegend, San Diego, CA, USA).
The second tube was to detect follicular T cells and included mAbs: CD4-APC-Vio770, CD45RA-PE-Vio770, CCR6-APC, CXCR3-VioBright FITC CXCR5 PerCP-Cy5.5, and CCR4-PE, all from Miltenyi Biotec (40133 Bologna, Italy).
The third tube was to phenotype regulatory T cells according to the BD Human Regulatory T Cell Cocktail (CD4-FITC (SK3), CD25-PECy7 (2A3), CD127 Alexa Fluor® 647, BD, San Jose, CA, USA), CXCR5 PerCP-Cy5, CD8 horizon450, and CD3 APC-Cy7 (OKT3) (Biolegend, San Diego, CA, USA).
Gating was carried out using Kaluza software 2.1 (Beckman Coulter, Inc., Danvers, MA, USA): details are shown in the Supplementary Materials. The T and B cell subsets are also described in the Supplementary Materials.
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4

Multiparametric Analysis of T-cell Subsets

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Foxp3-APC (ebioscience, clone 236A/E7) and mIgG1 κ APC isotype control (ebioscience, clone P3.6.2.8.1), CD25-PE (Miltenyi), CTLA-4-BV421 (BD Biosciences, clone BNI3) and mIgG2a κ BV421 isotype control (BD Biosciences), IFN-γ-PE and mIgG1 κ PE isotype control (ebioscience), IFN-γ-FITC and mIgG1 κ FITC isotype control (ebioscience), IL-17A eFluor450 and mIgG1 κ eFlour450 isotype control (ebioscience), CD45RA-PE-Vio770 (Miltenyi), CD45RA-FITC (Miltenyi), CD45RO-PE (BD Biosciences), CD4-PerCP (BD Biosciences), CD3-PE-Vio770 (Miltenyi), CD8-eFlour450 (ebioscience), fixable viability dye-eFlour780 (ebioscience), CFSE (Molecular Probes).
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5

Multiparametric Flow Cytometry Panel

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The following fluorochrome‐labeled monoclonal anti‐human antibodies were used to identify central and effector cells: CD3‐APC‐Cy7, CD4‐V450 (REA623), CD45RA‐PE‐Vio770 (REA562), CD196 (CCR6)‐APC (REA190), CD183 (CXCR3)‐VioBright FITC (REA232), CD194 (CCR4)‐PE (REA279) and PerCP‐Cy5.5 anti‐CXCR5 (all from Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were stained for 30 min at 4°C, measured with a FACSCanto II flow cytometer and analyzed using Kaluza Analysis version 2.1 (Beckman Coulter Life Sciences). Supporting information, Figure S3 reports the gating strategy for central and effector cell phenotyping panel, while Supporting information, Table S1 summarizes the main cell subsets identified from the analysis.
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