A minimum of 350,000 freshly isolated PBMCs were incubated with an Fc receptor-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and the cells were stained with fluorochrome-conjugated antibodies specific for the surface molecules of interest. For B cells the following antibodies were used: CD19 (PerCP/Cy5.5; HIB19), CD27 (FITC; 323), CD38 (BV421, HIT2) and CD11c (PE/Cy7; Bu15), all from BioLegend (San Diego, CA, USA). For T cells, the following antibodies were used: CD3 (APC/Cy7; HIT3a), CD4 (PerCP/Cy5.5; RPA-T4), CD25 (PE; M-A251), CD127 (APC; A019D5), CXCR5 (AF488; J252D4), PD-1 (BV605; EH12.2H7), CXCR3 (PE/Cy7; G025H7) and CCR6 (BV421; G034E3) from BioLegend. Where applicable, corresponding isotype controls were used. Absolute counts of T and B cell subpopulations were measured using TruCount beads (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry data acquisition was performed using a FACS Canto II flow cytometer (BD Biosciences), and data analysis was conducted using FlowJo software (Tree Star, Inc., Ashland, OR, USA).
Cd4 percp cy5.5 rpa t4
Manufactured by BioLegend
Sourced in United States
The CD4 PerCP/Cy5.5 (RPA-T4) is a fluorescently labeled antibody used for the identification and enumeration of CD4+ T cells in flow cytometric analysis. It binds to the CD4 surface antigen expressed on T helper cells. The PerCP/Cy5.5 fluorochrome allows for multicolor flow cytometric applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsr 2 cytometer, by BD (3 mentions)
Hiv p24 fitc kc57, by Beckman Coulter (3 mentions)
Cd3 pacific blue, by BD (3 mentions)
Cd3 pacific blue, by BioLegend (2 mentions)
Il 17a pe, by BioLegend (2 mentions)
Cd4 alexa fluor 700, by BioLegend (2 mentions)
Hiv p24 pe kc57, by Beckman Coulter (2 mentions)
Ccr6 pe, by BioLegend (2 mentions)
Cd45ra alexa efluor 780, by BioLegend (2 mentions)
Rorc2 alexa fluor 647, by BioLegend (2 mentions)
Ifn γ alexa fluor 700, by BioLegend (2 mentions)
Cd45ra alexa efluor 780, by BD (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Aria 2, by BD (2 mentions)
Cd19 percp cy5.5 hib19, by BioLegend (1 mentions)
Cd3 apc cy7 hit3a, by BioLegend (1 mentions)
Trucount beads, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
4 protocols using cd4 percp cy5.5 rpa t4
1
Comprehensive Immune Cell Profiling
2
Characterization of Th17 Cell Subsets
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The following fluorochrome-conjugated Abs were used for flow cytometry analysis: HIV-p24 FITC (KC57) (Beckman Coulter), HIV-p24 PE (KC57) (Beckman Coulter), CD3 Pacific blue (UCHT1), CD4 PerCP/Cy5.5 (RPA-T4) (BioLegend), CD4 Alexa Fluor 700 (RPA-T4), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), RORC2 Alexa Fluor 647 (Q31-378), Ki-67 BUV395 (B56), IL-17A PE (eBio64DEC17), and IFN-γ Alexa Fluor 700 (B27). The Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid, Life Technologies) was used to exclude dead cells. Intracellular staining was performed using the BD Cytofix/Cytoperm Kit (BD Biosciences), and intranuclear staining was performed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set. Cells were analyzed with the BD-LSRII cytometer, BD LSRFortessa and BD-Diva (BD Biosciences), and FlowJo version 10 (Tree Star, Inc.). The positivity gates were placed using fluorescence minus one strategy (8 (link), 20 (link)). For fluorescence-activated cell sorting (FACS), memory CD4 T cells from the PBMCs of ART+ PLWH were isolated by negative selection using magnetic beads. CCR6+RORC2+, CCR6+RORC2−, and CCR6−RORC− T cells were sorted by FACS (BDAria II; BD Biosciences) using the Abs CD3 Pacific blue (UCHT1), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), and RORC2 Alexa Fluor 647 (Q31-378).
3
Flow Cytometry Analysis of HIV-related Markers
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The following fluorochrome-conjugated Abs were used for flow cytometry analysis: HIV-p24 FITC (KC57) (Beckman Coulter, Fullerton, CA, USA), CD3 Pacific blue (UCHT1), CD3 Alexa Fluor 700 (UCHT1), BST2 BV421 (Y129) (BD Biosciences, San Diego, CA, USA) and CD4 PerCP/Cy5.5 (RPA-T4) (Biolegend, San Diego, CA, USA). BST2 staining was also performed with homemade primary rabbit polyclonal BST2 Abs (provided by Dr. Éric Cohen) and secondary goat anti-rabbit IgG conjugated with Alexa Fluor 594 (Abcam, Cambridge, UK). Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid, Life Technologies, Burlington, Ontario, CA) was used to exclude dead cells. Cells were analyzed with the BD-LSRII cytometer and BD-Diva (BD Biosciences) and FlowJo version 10 (Tree Star, Inc., Ashland Oregon, USA). The positivity gates were placed using fluorescence minus one (FMO) strategy (Roederer, 2002 (link)).
4
Comprehensive Flow Cytometry Profiling of HIV+ Cells
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The following fluorochrome-conjugated Abs were used for flow cytometry analysis: HIV-p24 FITC (KC57) (Beckman Coulter, Fullerton, CA, USA), HIV-p24 PE (KC57) (Beckman Coulter, Fullerton, CA, USA), CD3 Pacific blue (UCHT1), CD4 PerCP/Cy5.5 (RPA-T4) (Biolegend, San Diego, CA, USA), CD4 Alexa Fluor 700 (RPA-T4), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), RORC2 Alexa Fluor 647 (Q31-378), Ki-67 BUV395 (B56), IL-17A PE (eBio64DEC17) and IFN-γ Alexa Fluor 700 (B27). Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid, Life Technologies, Burlington, Ontario, CA) was used to exclude dead cells. Intracellular staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences) and intranuclear staining was performed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set. Cells were analysed with the BD-LSRII cytometer, BD LSRFortessa and BD-Diva (BD Biosciences) and FlowJo version 10 (Tree Star, Inc., Ashland Oregon, USA). The positivity gates were placed using fluorescence minus one (FMO) strategy (8, 20) . For FACS, memory CD4 T cells from PBMCs of ART+ PLWH were isolated by negative selection using magnetic beads. CCR6 + RORC2+, CCR6+RORC2-and CCR6 -RORC-T cells were sorted by FACS (BDAria II; BD Biosciences) using the antibodies CD3 Pacific blue (UCHT1), CCR6 PE (11A9), CD45RA Alexa eFluor 780 (HI100), and RORC2 Alexa Fluor 647 (Q31-378).
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