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2 protocols using hucd2 pe

1

Multiparametric Flow Cytometry Analysis

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Cells were first stained with LiveDead aqua or blue (Life Technologies). Following FcR-Block (2.4G2), cells were stained using the following monoclonal antibodies (all used at 1:200 dilution): CD3-APCe780, CD4-APC or A700, CD8α-PE/Cy7, CD11c-APC or BV421, MHC-II-PerCP/Cy5.5 or eFlour450, CD11b-BV711 or PE, CD19-e780, CD80-PerCP/Cy5.5, CD40-PE, CD44-BV570, CD69-FITC CD86-AF488, Foxp3-e450, F4/80-PE/Cy7, Gr1-FITC, HuCD2-PE, IgM-APCe780, SiglecF-PE and TCRβ-APCe780 (BD Biosciences, BioLegend or eBioscience). Foxp3 staining was performed with the eBioscience FoxP3 staining kit. Samples were acquired using FACS LSR II or FACS Canto II using BD FACSDiva software and analysed with FlowJo v.9 software (Tree Star). Cytokines were measured in culture supernatants by ELISA using paired monoclonal antibody, and recombinant cytokine standards, or Duosets (eBioscience, BD Biosciences, BioLegend, R&D Systems and Peprotech).
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2

Flow Cytometry Immunophenotyping Panel

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Cells were first stained with LiveDead aqua or blue (Life Technologies). Following FcR-Block (2.4G2), cells were stained using the following mAb (all used at 1:200 dilution): CD3-APCe780, CD4- APC or A700, CD8α-PE/Cy7, CD11c- APC or BV421, MHCII-PerCP/Cy5.5 or eFlour450, CD11b-BV711 or PE, CD19-e780, CD80-PerCP/Cy5.5, CD40-PE, CD44-BV570, CD69-FITC CD86-AF488, Foxp3-e450, F4/80-PE/Cy7, Gr1-FITC, HuCD2-PE, IgM-APCe780, SiglecF-PE and TCRβ-APCe780 (BD Biosciences, BioLegend, or eBioscience). Foxp3 staining was performed with the eBioscience FoxP3 staining kit. Samples were acquired using FACS LSR II or FACS Canto II using BD FACSDiva software and analyzed with FlowJo v.9 software (Tree Star). Cytokines were measured in culture supernatants by ELISA using paired mAb, and recombinant cytokine standards, or Duosets (eBioscience, BD Biosciences, BioLegend, R&D Systems, and Peprotech).
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