PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4+/CD8+ T cell ratio (Supplementary Table 1 ). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.
Ccr4 pe
Manufactured by BioLegend
CCR4-PE is a fluorochrome-conjugated antibody that binds to the chemokine receptor CCR4. CCR4 is expressed on the surface of various immune cell types, including T helper 2 (Th2) cells, regulatory T cells (Tregs), and certain subsets of natural killer (NK) cells. The PE (phycoerythrin) fluorochrome allows for the detection and analysis of CCR4-expressing cells using flow cytometry.
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Lab products found in correlation
Cd3 fitc, by BD (3 mentions)
Cd8 bv510, by BioLegend (2 mentions)
Cd28 bv421, by BioLegend (2 mentions)
Cd45ro fitc, by BioLegend (2 mentions)
Cd45ra apc, by BioLegend (2 mentions)
Cd45ra apc cy7, by BioLegend (2 mentions)
Cd138 bv421, by BioLegend (2 mentions)
Ccr6 bv421, by BioLegend (2 mentions)
Il 17 bv786, by BioLegend (2 mentions)
Ifn γ pe cy7, by BioLegend (2 mentions)
Cd38 pe cy7, by Beckman Coulter (2 mentions)
Cxcr3 pe, by BioLegend (2 mentions)
Cd27 pe, by BioLegend (2 mentions)
Cd4 apc cy7, by BioLegend (2 mentions)
Ly6g fitc, by BD (2 mentions)
Cd62l pe, by BD (2 mentions)
Ly6c pe, by BD (2 mentions)
Cxcr3 pe cy7, by BioLegend (2 mentions)
Ccr6 apc, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
6 protocols using ccr4 pe
1
Multicolor Flow Cytometry for T and B Cell Subsets
2
Characterization of CAR-T Cells by Flow Cytometry
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The following antibodies were used: CD3 (VioBlue; Miltenyi Biotech or Pacific blue and PE; BioLegend), CD4 (FITC or APC-Cy7; BioLegend), CD8 (PE-Cy7; BioLegend), CD3/CD19 antibodies (FITC/PE; BD), CD28 (PerCP-Cy5.5; eBioscience), PD-1 (FITC; clone: EH12.2H7; BioLegend), TIM-3 (APC-Cy7; BioLegend), LAG-3 (VioBlue; Miltenyi Biotech), CD45RA (APC-Vio770; Miltenyi Biotec or Brilliant Violet; BioLegend), CCR7 (PerCP-Vio770; Miltenyi Biotec or PerCP; BioLegend), CCR2 (APC; Biolegend), CCR4 (PE; Biolegend), CCR5 (Alexa Influenza 488; Biolegend), CXCR2 (PE-Cy7; Biolegend) and CXCR3 (FITC; Biolegend).
Transduction efficacy was determined on day 6 and day 9 of culture by labeling CAR-T cells with biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody (anti-Fab, Jackson Immunoresearch, West Grove, Pennsylvania) and streptavidin (APC conjugated; BioLegend). CD3+F(ab)2+ cells were defined as CAR-T cells. Isotype labeled cells (Jackson Immunoresearch) and untransduced cells served as negative controls. For further characterization, cells were stained with antibodies mentioned above. Cells were washed and re-suspended in cell staining buffer (BioLegend), incubated for 30 min with the antibodies on ice, washed and measured using MACSQuant FACS cytometer (Miltenyi Biotec). Samples were analyzed by FlowJo software (FlowJo LLC, Ashland, Oregon).
Transduction efficacy was determined on day 6 and day 9 of culture by labeling CAR-T cells with biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody (anti-Fab, Jackson Immunoresearch, West Grove, Pennsylvania) and streptavidin (APC conjugated; BioLegend). CD3+F(ab)2+ cells were defined as CAR-T cells. Isotype labeled cells (Jackson Immunoresearch) and untransduced cells served as negative controls. For further characterization, cells were stained with antibodies mentioned above. Cells were washed and re-suspended in cell staining buffer (BioLegend), incubated for 30 min with the antibodies on ice, washed and measured using MACSQuant FACS cytometer (Miltenyi Biotec). Samples were analyzed by FlowJo software (FlowJo LLC, Ashland, Oregon).
3
Immunophenotypic Analysis of Splenocytes
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Immunophenotypic analyses of splenoctyes from animals were assessed by flow cytometry. Antibodies to stain for MDSC were CD11b-APC (Clone M1/70; BD Biosciences), Ly6G-FITC (Clone 1A8; BD Biosciences), Ly6C-PE (Clone AL-21; BD Biosciences); for dendritic cells were CD11c (Clone HL3; BD Biosciences); for B cells B220-APC (Clone RA3–6B2; BD Biosciences), CD3-FITC (Clone 145–1011; BD Biosciences). T regulatory cells with a phenotype of CD4+CD25+FoxP3+ were evaluated using a commercially available kit (eBiosciences, San Diego, CA). For T cell activation markers, cells were stained with antibodies specific for CD4-PE-Cy7 (Clone RM4–5; BD Biosciences), CD8-PE-Cy7 (Clone 53–6.7; BD Biosciences), CD62L-PE (Clone MEL-14; BD Biosciences), and CD44-Bv650 (Clone IM7; Biolegend). To determine Th1 and Th2 phenotypes, cells were stained using fluorochrome conjugated antibodies targeted CXCR3-PE-Cy7 (Clone CXCR13–173; Biolegend), CCR4-PE (Clone 2G12; Biolegend), and CCR6-APC (Clone CK4-L3; BD Biosciences). Cells were incubated on ice for 30 minutes, washed, and fixed in PBS containing 1% formalin for flow cytometric analysis on a LSRII flow cytometer (BD Biosciences).
4
Tumor-Infiltrating Immune Cell Profiling
5
Multicolor Flow Cytometry of PBMC
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We isolated peripheral blood mononuclear cells (PBMC) from peripheral blood by Ficoll gradient and we stored them in liquid nitrogen for batched analysis. For multicolor flow cytometry analyses, we used the following monoclonal antibodies: from Becton Dickinson (BD, Franklin Lakes, NJ), CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CD71-PE, IgD-PerCP-Cy5.5, CD25-APC-Cy7, CD138-BV421, CCR4-PE, CD27-PE, CD95-BV421, CD28-BV421, TIM3-BV421, CCR6-BV421, CCR7-AF700, CXCR3-PE, IL-2-PE, IL-17-BV786, and IFN-γ-PE-Cy7; from Biolegend (San Diego, CA), CD19-BV510, CD56-FITC, CD27-APC, CD127-FITC, CD57-PerCp-Cy5.5, PD-1-APC-Cy7, CXCR5-FITC, LAG3-APC, IL-10-PerCP-Cy5.5, and TNF-α-FITC. From eBioscience (San Diego, CA), CD4-PE-Cy7, CD21-PE, and CD24-APC-Cy7 were used. From Miltenyi Biotec (Auburn, CA), CD25-APC and anti-KLRG1-PE were used. From Beckman Coulter (Indianapolis, IN), CD38-PE-Cy7 was used.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
We performed intracellular staining for IL-2, IL-4, IL-17, IFN-g, and TNF-a together with extracellular markers for CD4+, CD8+, and CD19+. Cells were fixed and permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions.
Data were acquired (> 1 × 106 events) on a 3-laser FACSLyric flow cytometer (BD Biosciences) and analyzed with FlowJo® software.
6
Isolation of Murine Cardiac T Cells
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To isolate T cells from heart tissue, native or transplanted murine hearts were harvested and cut into small pieces. The tissue fragments were digested in 1 ml Hanks medium, containing 608 U/ml Collagenase I (Sigma), 187.5 U/ml Collagenase XI (Sigma), 90 U/ml Hyaluronidase (Sigma), and 90 U/ml DNase (Sigma), for 1 hr at 37°C with agitation. Digested samples were passed through a 70 μm cell strainer to obtain a single-cell suspension and were then washed in FACS buffer before cell surface staining with a 1:200 dilution of conjugated antibodies against CCR4-PE (Biolegend), CXCR3-PerCP (Biolegend), CD4-PECy7 (Biolegend), CD8-APC (Biolegend), and CD44-af450 (eBioscience). Cells were incubated for 1 hr at 4°C before being washed twice in FACS buffer and then fixed with 200 μl Fixation Buffer (eBioscience) for 1 hr at 4°C. Cells were washed twice with Fixation/Permeabilisation buffer before intracellular staining with a 1:200 dilution of FITC-conjugated antibody against c-Met (eBioscience). Cells were incubated for 1 hr at 4°C before being washed twice in Fixation/Permeabilisation buffer and being analyzed on a FACS Fortessa (BD).
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