Ccr4 percp cy5

Manufactured by BD

The CCR4 PercP-Cy5.5 is a laboratory equipment product made by BD. It is a fluorescently labeled antibody that targets the CCR4 receptor, which is expressed on certain immune cells. The PercP-Cy5.5 fluorescent label allows for the detection and analysis of CCR4-expressing cells using flow cytometry or other compatible instruments.

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3 protocols using ccr4 percp cy5

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.

Ramwadhdoebe T.H., Hähnlein J., van Kuijk B.J., Choi I.Y., van Boven L.J., Gerlag D.M., Tak P.P, & van Baarsen L.G. (2016). Human lymph-node CD8+ T cells display an altered phenotype during systemic autoimmunity. Clinical & Translational Immunology, 5(4), e67-.

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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.

Ramwadhdoebe T.H., Hähnlein J., Maijer K.I., van Boven L.J., Gerlag D.M., Tak P.P, & van Baarsen L.G. (2016). Lymph node biopsy analysis reveals an altered immunoregulatory balance already during the at‐risk phase of autoantibody positive rheumatoid arthritis. European Journal of Immunology, 46(12), 2812-2821.

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Multicolor Flow Cytometry Analysis of PBMC

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PBMC samples were thawed in 37°C RPMI medium 1640 + GlutaMAX (Life Technologies) and thereafter washed in RPMI and stained in PBS containing 0.5% bovine serum albumin. For phenotyping of CD3+ T-cells, the following antibodies were used: CD45RA-FITC, CD62L-PE, CCR7-PE-CY7, CD3-APC, CD8-BV421, CD4-HV510 (BD Biosciences), CD27-PerCP (Nordic Biosite). Natural Killer cells, B-cells, and γ/δ cells were stained with the following antibodies: CD16-FITC, CD56-PE, CD19-PE-CY7, CD3-APC (BD Biosciences), and γ/δ -BV421 (Nordic Biosite). Myeloid derived suppressor cells were stained with: CD33-FITC, HLA-DR-PerCP, lineage = CD3-, CD19-, and CD56-PE-Cy7, CD11b-APC (BD Biosciences), CD14-BV421 (Nordic Biosite). Regulatory T-cells were stained with CD45RA-FITC, CCR4-PerCP-Cy5.5, CD127-PE-Cy7, CD4-APC, CD25-BV421 (BD Biosciences), FoxP3-PE (eBiosciences). Dead cell marker APC-Cy7 near IR (Invitrogen) fluorescent reactive dye was used to exclude dead cells. For intracellular staining of transcription factor FoxP3, we used Transcription Factor Staining Buffer set (eBioscience) according to guidelines issued by the manufacturer.

Kjeldsen J.W., Iversen T.Z., Engell-Noerregaard L., Mellemgaard A., Andersen M.H, & Svane I.M. (2018). Durable Clinical Responses and Long-Term Follow-Up of Stage III–IV Non-Small-Cell Lung Cancer (NSCLC) Patients Treated With IDO Peptide Vaccine in a Phase I Study—A Brief Research Report. Frontiers in Immunology, 9, 2145.

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