Multiparametric flow cytometry analysis

Single cell suspensions of T cells were stained as described (24 (link)) with a fluorochrome-labeled mAb mixture directed against the cell surface markers CD3, CD4, CD25, CD39, CD44, CD45RA, CD62L, CD95, CD127, CCR7 and CXCR3 (BD PharMingen, Franklin Lakes, NJ, or BioLegend, San Diego, CA). Intracellular B-cell lymphoma 2 (Bcl-2), Foxp3, the proliferation markerKi67, cytotoxic T lymphocyte Ag-4 (CTLA-4) and Helios were stained using an eBioscience Foxp3 Staining kit, according to the manufacturer's instructions. Apoptosis was determined by Annexin V staining (BD PharMingen) and membrane integrity by propidium iodide (PI) staining. Data were acquired on a LSR II or LSR Fortessa (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

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Zhang H., Guo H., Lu L., Zahorchak A.F., Wiseman R.W., Raimondi G., Cooper D.K., Ezzelarab M.B, & Thomson A.W. (2015). Sequential monitoring and stability of ex vivo-expanded autologous and non-autologous regulatory T cells following infusion in non-human primates. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 15(5), 1253-1266.

Publication 2015

CD45RA CD62L CD127 CXCR3 Foxp3 Staining kit Annexin V LSR Fortessa

Annexin v Apoptosis B cell lymphoma Cd25 Cd44 Cd62l Cell Cxcr3 Cytotoxic t lymphocyte Fluorochrome Intracellular Membrane Propidium iodide T cells Tree

Corresponding Organization : University of Pittsburgh

Other organizations : University of Wisconsin–Madison

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Variable analysis

independent variables

Fluorochrome-labeled monoclonal antibodies (mAbs) directed against cell surface markers: CD3, CD4, CD25, CD39, CD44, CD45RA, CD62L, CD95, CD127, CCR7, and CXCR3

dependent variables

Expression of intracellular markers: B-cell lymphoma 2 (Bcl-2), Foxp3, Ki67, cytotoxic T lymphocyte Ag-4 (CTLA-4), and Helios
Apoptosis determined by Annexin V staining
Membrane integrity determined by propidium iodide (PI) staining

control variables

Single cell suspensions of T cells

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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