After stimulation, cells were washed, stained with LIVE/DEAD® Fixable Near-IR Stain (Invitrogen) and, subsequently, surface stained with the following antibodies: CD14-APC/Alexa Fluor 750 (Invitrogen) and CD19-APC/Alexa Fluor 750 (Invitrogen), CD4-FITC (BD), CD27-BV711 (BD), CCR4-BV510 (Biolegend), CCR6-BV605 (Biolegend), CXCR3-PE-Cy7 (BD Biosciences), KLRG1-PerCP/eFluor 710 (eBioscience), and HLA-DR-PE (BD). Cells were then fixed and permeabilized using Cytofix/Cytoperm buffer (BD) and stained with CD3-BV650 (BD), IL-2-PE/Dazzle™ (Biolegend), TNFα-eFluor 450 (eBioscience), and IFNγ-Alexa Fluor 700 (BD). Finally, cells were washed and fixed in 1% formaldehyde in PBS. Samples were acquired on a LSR-II (BD) and analyses were performed using FlowJo (Treestar). A positive IFNγ response was defined as at least twice the background measured in the presence of co-stimulatory antibodies without antigen. Cell polyfunctionality was analyzed using Pestle and Spice software (19 (link)). The gating strategy is presented in Figure S1 in Supplementary Material.
Cxcr3 pe cy7
Manufactured by BD
CXCR3-PE-Cy7 is a fluorescently-labeled antibody that binds to the CXCR3 receptor, which is expressed on various immune cells. The PE-Cy7 conjugate allows for the detection and analysis of CXCR3-positive cells using flow cytometry.
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Lab products found in correlation
Live dead fixable near ir stain, by Thermo Fisher Scientific (1 mentions)
Cd14 apc alexa fluor 750, by Thermo Fisher Scientific (1 mentions)
Cd19 apc alexafluor750, by Thermo Fisher Scientific (1 mentions)
Bv605 ccr6, by BioLegend (1 mentions)
Cd3 bv650, by BD (1 mentions)
Tnfα efluor 450, by Thermo Fisher Scientific (1 mentions)
Ifnγ alexa fluor 700, by BD (1 mentions)
Hla dr pe, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Integrin β7 fitc clone fib504, by Thermo Fisher Scientific (1 mentions)
Software, by FlowJo (1 mentions)
Aria 2 flow cytometer, by BD (1 mentions)
Dasatinib, by Bristol-Myers Squibb (1 mentions)
2 protocols using cxcr3 pe cy7
1
Multi-parametric Phenotyping of Immune Cells
2
Characterization of OmpC-specific CD4+ T cells
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Frozen PBMCs from CD patients and HC donors were thawed and incubated at 37°C with 1 μmol/L dasatinib (Bristol-Myers Squibb, New York, NY) for 8 minutes to prevent TCR down-regulation. Samples then were incubated with OmpC–PE–labeled tetramer for 90 minutes at room temperature. Tetramer enrichment was performed using the Miltenyi (Cologne, Germany) OctoMACS separation system with anti-PE beads. A total of 50 μL from the pre-enrichment samples was obtained to calculate tetramer frequency (described later). Samples were stained with appropriate monoclonal antibody mixtures (integrin β7-FITC [clone FIB504; eBioscience, San Diego, CA], CD127-BV650 [clone A019D5; eBioscience], CD4-APC-e780 [clone RPA-T4; eBioscience], Viaprobe-PerCP-Cy5.5 [Via-Probe Cell Viability Solution; BD Biosciences, Franklin Lakes, NJ], CD49d-BV510 [clone 9F10; BD Biosciences], CD45RA-BV605 [clone HI100; BD Biosciences], CCR7-A700 [clone 150503; BD Biosciences], CXCR3-PE-Cy7 [clone 1C6/CXCR3; BD Biosciences], CD14-PerCP-Cy5.5 [clone HCD14; BioLegend, San Diego, CA], CD161-BV421 [clone HP-3G10; BioLegend], CD19-PerCP-Cy5.5 [clone HIBI9; BioLegend], CD25-APC [clone BC96; BioLegend], and tetramer-OmpC-PE). Live CD4+, CD45RA-, OmpC-specific T cells were identified and characterized on an Aria II flow cytometer (BD Biosciences) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR).
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