Multicolor Flow Cytometry Chimerism Analysis

Multicolor flow-cytometric analysis of multilineage macrochimerism and Vβ-subunit expression were performed as described previously.5 (link), 13 (link) Chimerism was calculated as the net percentage of donor MHC Class I⁺ (H-2D^d, 34-2-12) cells among specific leukocyte lineages, as described previously.5 (link), 13 (link) Mice were considered chimeric if donor cells were detectable by flow cytometry within both the myeloid lineage and at least one lymphoid lineage. For analysis of Treg phenotype, MAbs with specificity against CD4 (RM4-4) and CD25 (7D4) were used. For intracellular staining, a FoxP3 (FJK-16s) staining kit (eBioscience) was used according to the manufacturer’s protocol. Propidium iodide (PI) was used for dead cell exclusion when appropriate. Surface staining was performed according to standard procedures and flow-cytometric analysis was done on a Coulter Cytomics FC 500 System using CXP software (Coulter, Austria) for acquisition and analysis.

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Pilat N., Farkas A.M., Mahr B., Schwarz C., Unger L., Hock K., Oberhuber R., Aumayr K., Wrba F, & Wekerle T. (2014). T-regulatory cell treatment prevents chronic rejection of heart allografts in a murine mixed chimerism model. The Journal of Heart and Lung Transplantation, 33(4), 429-437.

Publication 2014

FoxP3 (FJK-16s) staining kit

Cd25 Cell Cells leukocyte Chimeric Chimerism Donor Flow cytometric Intracellular Lymphoid Mabs Mhc class i Mice Phenotype Propidium iodide Subunit

Corresponding Organization :

Other organizations : Medical University of Vienna, Innsbruck Medical University

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Variable analysis

independent variables

Multicolor flow-cytometric analysis of multilineage macrochimerism
Vβ-subunit expression

dependent variables

Chimerism (net percentage of donor MHC Class I+ cells among specific leukocyte lineages)
Treg phenotype (CD4, CD25, FoxP3)

control variables

Propidium iodide (PI) for dead cell exclusion
Surface staining performed according to standard procedures
Flow-cytometric analysis done on a Coulter Cytomics FC 500 System using CXP software

controls

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

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