Mononuclear cells were isolated from rectum and axillary LNs as described previously (20 (link), 21 (link), 23 ). Whole blood and mononuclear cells from tissues were stained with LIVE/DEAD Near-IR Dead Cell stain (Life Technologies) and antibodies specific to CD3, CD4, CD8, PD-1, HLA-DR, Gag-CM9, CD28, and CD95. To stain cells with intracellular markers, cells were fixed and permeabilized with the eBioscience FoxP3/Transcription Factor staining buffer set following the manufacturer’s instructions and stained with antibodies specific to FoxP3, Ki67, GrzB, and perforin. These samples were acquired on an LSR Fortessa (BD Biosciences).
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Mononuclear Cell Isolation and Staining
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Antibodies
Axillary
Blood
Buffer
Cells
Hla dr
Intracellular
Perforin
Rectum
Tissues
Transcription factor
Corresponding Organization : Emory University
Other organizations : Dana-Farber Cancer Institute, Harvard University
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Variable analysis
independent variables
- Tissue source of mononuclear cells (rectum and axillary LNs)
- Expression of CD3, CD4, CD8, PD-1, HLA-DR, Gag-CM9, CD28, CD95, FoxP3, Ki67, GrzB, and perforin in mononuclear cells
- Isolation of mononuclear cells from tissues as described previously (20, 21, 23)
- Staining of whole blood and mononuclear cells with LIVE/DEAD Near-IR Dead Cell stain and antibodies
- Fixation and permeabilization of cells for intracellular staining using eBioscience FoxP3/Transcription Factor staining buffer set
- Positive controls: Not specified
- Negative controls: Not specified
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