Fourteen-parameter flow cytometric analysis was performed on peripheral blood, LN, and RB derived cells according to standard procedures using a panel of monoclonal antibodies that we and others have shown to be cross-reactive with SMs and RMs.12 (link) The following antibodies were used at predetermined optimal concentrations: anti-CCR6-PE-Cy7 (clone 11A9), anti-CCR5-PE and -APC (clone 3A9), anti-CD3-APC-Cy7 (clone SP34-2), anti-CD62L-FITC (clone SK11), anti-CD95-PE-Cy5 (clone DX2), anti-Ki-67-Alexa700 (clone B56), anti-IFN-γ-Alexa700 (clone B27), and anti-CXCR3-AlexaFluor 488 (clone 1C6/CXCR3) from BD Biosciences; anti-CD161-PE (clone HP-3G10) and anti-IL-17-Alexa488 (clone eBio64DEC17) from eBioscience; anti-CD28-ECD (clone CD28.2) from Beckman Coulter; anti-CD4-BV421 (clone OKT4), anti-CD4-BV605 (clone OKT4), and anti-CD161-BV421 (clone HP-3G10) from Biolegend; anti-CD8-Qdot705 (clone 3B5) and Aqua Live/Dead amine dye-AmCyan from Invitrogen. Flow cytometric acquisition was performed on at least 100,000 CD3+ T cells on an LSRII cytometer driven by the FACS DiVa software, or at least 10,000 CD3+ T cells for rectal biopsy-derived cells. The data acquired were analyzed using FlowJo software (version 9.8.5; TreeStar).
Protocol detail
Comprehensive Immune Profiling by Flow Cytometry
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anti-CXCR3-AlexaFluor 488 (clone 1C6/CXCR3)
anti-CD161-PE (clone HP-3G10) and anti-IL-17-Alexa488 (clone eBio64DEC17)
anti-CD28-ECD (clone CD28.2)
anti-CD161-BV421 (clone HP-3G10)
anti-CD8-Qdot705 (clone 3B5)
Aqua Live/Dead amine dye-AmCyan
Amine
Anti cd3
Antibodies
Biopsy
Blood
Ccr5
Ccr6
Cd161
Cd62l
Cells
Clone
Cross reactive
Cxcr3
Fitc
Flow cytometric
Ifn γ
Il 17
Monoclonal antibodies
Rectal
T cells
Corresponding Organization :
Other organizations : Emory University, Tulane University, Brigham and Women's Hospital, Ragon Institute of MGH, MIT and Harvard, Georgia Department of Public Health
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Variable analysis
independent variables
- None explicitly mentioned
- Frequency of T cell subsets (e.g., CCR6+, CCR5+, CD62L+, CD95+, Ki-67+, IFN-γ+, IL-17+, CXCR3+, CD161+) from peripheral blood, lymph node, and rectal biopsy samples
- Monoclonal antibodies used at predetermined optimal concentrations
- Flow cytometric acquisition of at least 100,000 CD3+ T cells for peripheral blood and lymph node samples, and at least 10,000 CD3+ T cells for rectal biopsy samples
- Flow cytometric data analyzed using FlowJo software (version 9.8.5)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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