Cultured T cells were washed in PBS/0.1% BSA and re-suspended at 1×106 cells in 50 uL Brilliant Buffer (BD Biosciences) supplemented with 4% rat serum for 15 minutes at 4C. Cells were then incubated for 30 minutes at 4°C in 100 μL of Brilliant Buffer using the following antibody fluorophore conjugates (all from BD Biosciences unless otherwise noted): CD7 BV421, CD4 BV510, CCR4 BV605 (BioLegend), CD8 BV650, CD196 BV786 (BioLegend), CD3 AF488, CD45RA PerCPCy5.5, CD183 PE, CD197 PE-CF594, CD185 PE-Cy7 (BioLegend), and CCR10 APC (R&D Systems). Full details of fluorophore conjugated antibodies can be found in supplemental table 5 . Cells were then washed twice in PBS/0.1% BSA and data acquired on a ZE5 (Yeti) cytometer (BioRad/Propel Labs). Compensation and analyses were performed on FlowJo V10 (TreeStar) using fluorescence minus one (FMO) controls. Statistical analyses were performed on GraphPad Prism 7 using 2-way ANOVA with Bonferroni post-hoc corrections.
Ccr4 bv605
Manufactured by BioLegend
The CCR4 BV605 is a fluorochrome-conjugated monoclonal antibody that binds to the chemokine receptor CCR4 expressed on the surface of certain cell types, such as T helper 2 (Th2) cells. It is designed for use in flow cytometry applications to identify and quantify CCR4-positive cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Brilliant buffer, by BD (2 mentions)
Cd4 bv510, by BioLegend (2 mentions)
Cd8 bv650, by BioLegend (2 mentions)
Cd3 af488, by BioLegend (2 mentions)
Cd45ra percp cy5, by BioLegend (2 mentions)
Cd183 pe, by BioLegend (2 mentions)
Ccr10 apc, by R&D Systems (2 mentions)
Cd7 bv421, by BioLegend (2 mentions)
Cd196 bv786, by BioLegend (2 mentions)
Cd197 pe cf594, by BioLegend (2 mentions)
Cd185 pe cy7, by BioLegend (2 mentions)
Cd25 pe, by BioLegend (1 mentions)
Foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Cd127 af488, by BioLegend (1 mentions)
Ctla 4 pe cf594, by BD (1 mentions)
Cd4 percp, by BD (1 mentions)
Cd45ra af700, by BD (1 mentions)
Ccr6 bv421, by BioLegend (1 mentions)
Cxcr3 fitc, by BioLegend (1 mentions)
Cd14 percpcy5, by Thermo Fisher Scientific (1 mentions)
4 protocols using ccr4 bv605
1
Multiparameter Flow Cytometry of T Cells
2
Phenotyping Treg Subsets in PBMC
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Frozen PBMC from WNV infected subjects were thawed, rested for 30 minutes and then co-stained with antibodies for surface markers and cytokines of interest, including CD4 PerCP (BD, clone RPA-T4), CD25 PE (BioLegend, clone BC96), CD127 AF488 (Biolegend, clone 8019D5), FoxP3 APC (eBioscience, clone PCH101), CTLA-4 PE-CF594 (BD, clone BN13) and CCR4 BV605 (Biolegend, clone L29IH4). After 20 min at 4 degrees, cells were washed and immediately analyzed by flow cytometry.
3
Multiparameter Flow Cytometry of T Cells
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Cultured T cells were washed in PBS/0.1% BSA and re-suspended at 1×106 cells in 50 uL Brilliant Buffer (BD Biosciences) supplemented with 4% rat serum for 15 minutes at 4C. Cells were then incubated for 30 minutes at 4°C in 100 μL of Brilliant Buffer using the following antibody fluorophore conjugates (all from BD Biosciences unless otherwise noted): CD7 BV421, CD4 BV510, CCR4 BV605 (BioLegend), CD8 BV650, CD196 BV786 (BioLegend), CD3 AF488, CD45RA PerCPCy5.5, CD183 PE, CD197 PE-CF594, CD185 PE-Cy7 (BioLegend), and CCR10 APC (R&D Systems). Full details of fluorophore conjugated antibodies can be found in supplemental table 5 . Cells were then washed twice in PBS/0.1% BSA and data acquired on a ZE5 (Yeti) cytometer (BioRad/Propel Labs). Compensation and analyses were performed on FlowJo V10 (TreeStar) using fluorescence minus one (FMO) controls. Statistical analyses were performed on GraphPad Prism 7 using 2-way ANOVA with Bonferroni post-hoc corrections.
4
Quantifying Antigen-Specific T-Cell Frequencies
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Analysis of T-cell frequency was accomplished using our previously published approach (26 (link)). Briefly, 40–60 × 106 PBMCs were resuspended in a total of 400–600 µL media, divided into two or three independent tubes of 20 × 106 cells (200 µL) each, incubated with 50 nmol/L dasatinib for 10 min at 37°C, and stained with 20 µg/mL PE-labeled, PE-CF594–labeled, or PE-Cy5–labeled HIP tetramers at room temperature for 120 min (three tetramers per tube for a total of six hybrid peptide tetramers plus an nonimmunogenic control tetramer if adequate cells were available for a third staining tube). Cells were washed, incubated with PE-magnetic beads (Miltenyi Biotec) for 20 min at 4°C, and magnetically enriched; 1% of the cells were retained as a nonenriched sample. Enriched (bound) and nonenriched (precolumn) samples were stained with CD4 V500, CD14 PerCP-Cy5.5, and CD19 PerCP-Cy5.5 (eBioscience) and CD45RA AF700 (BD), CXCR3 FITC, CCR6 BV421, and CCR4 BV605 (BioLegend) for 15 min at 4°C. After washing, cells were labeled with ViaProbe (BD Biosciences) and analyzed on a FACSCanto (BD Biosciences), gating on CD4+CD14−CD19−ViaProbe− cells and plotting tetramer versus CD45RA. Frequencies were calculated as previously described (26 (link)).
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