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2 protocols using cd69 percp

1

Flow Cytometry Panel for Immune Cell Profiling

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Cells were stained for 30 min at 4 °C in PBS containing 0.01% NaN3 and 0.5% BSA with directly labelled antibodies against: CXCR3 alexa fluor488, CCR5 PE, CCR4 PercP-Cy5.5, CCR7 PE-Cy7, CCR6 alexa647, CD4 APC-H7,CD8 V500, CD3 V500, CD69 PercP, CD45 V500, Foxp3 PercP-Cy5.5, Granzyme-A PE, CD8 V450 (all from BD Biosciences), CD3 FITC (Sanquin, Amsterdam, The Netherlands), CD4 PE-Cy7, CD28 APC, CD8 APC-efluor780, CD45RA efluor450, CD45RO PE (all from eBioscience Inc., San Diego, CA, USA), Granzyme-K Fitc (Immunotools, Friesoythe, Germany), Granzyme-B APC (Invitrogen, Thermo Fisher Scientific Inc.) and Perforin PercP-Cy5.5, IL-10 Pe-Cy7 (Biolegend, San Diego, CA, USA). For cytokine staining, we used the Th1/Th2/Th17 kit from BD Biosciences. Cells were analysed on an FACS Canto II (BD Biosciences) and data were analysed using the FlowJo software (FlowJo, Ashland, OR, USA).
Data were plotted as frequency of positive cells or as the gMFI to illustrate cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest was normalized to the gMFI of the negative population.
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2

Multiparametric Flow Cytometry Immunophenotyping

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Cells were stained for 30 min at 4°C in PBS containing 0.01% NaN3 and 0.5% BSA with directly labeled antibodies against: CXCR3 Alexa fluor488, CCR5 PE, CCR4 PerCP‐Cy5.5, CCR7 PE‐Cy7, CCR6 Alexa647, CD4 APC‐H7, CD3 V500, CD69 PerCP, CD45 V500 (all from BD Biosciences); CD3 FITC (Sanquin); CD4 PE‐Cy7, CD45 RA eFluor450, and CD45RO PE (all from eBioscience Inc., San Diego, CA). For intracellular cytokine staining, we used IL‐10 PE‐Cy7 (BioLegend, San Diego, CA, USA) and the Th1/Th2/Th17 kit from BD Biosciences. For intracellular staining, after cell surface staining, cells were washed and fixed with FACS Cytofix fixation buffer (BD Biosciences). Following permeabilization (FACS Perm/Wash buffer [BD Biosciences]) cells were stained with markers as indicated. Cells were analyzed on a FACS Canto II (BD Biosciences). CS&T beads were run daily and the same machine with dedicated cytometer configuration was used for the measurements of the samples throughout the study with regular control runs to check compensation settings. Data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
Data were plotted as frequency of positive cells or as the gMFI for cytokine expression levels. To correct for experimental variation, the gMFI of cells of interest were normalized to the gMFI of the cytokine negative population.
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