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Purpose:
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A multicolor flow cytometry panel was designed for mouse (C57BL/6J) spleen and optimized to define the following nine T cell subsets: Treg (CD3+ CD4+ CD8— FoxP3+), CD4+ T naïve (CD3+ CD4+ CD8—FoxP3— CD44int/low CD62L+), CD4+ T central memory (CD3+ CD4+ CD8— FoxP3— CD44high CD62L+), CD4+ T effector memory (CD3+ CD4+ CD8— FoxP3— CD44high CD62L—), CD4+ T EMRA (CD3+ CD4+ CD8— FoxP3— CD44int/low CD62L—), CD8+ T naïve (CD3+ CD8+ CD4—CD44int/low CD62L+), CD8+ T central memory (CD3+ CD8+ CD4— CD44high CD62L+), CD8+ T effector memory (CD3+ CD8+ CD4— CD44high CD62L—), and CD8+ T EMRA (CD3+ CD8+ CD4— CD44int/low CD62L—). In each T cell subset, Ki-67 expression and DNA content were employed to distinguish the following cell cycle phases: G0 (Ki67—, with 2n DNA), G1 (Ki67+, with 2n DNA), and S-G2/M (Ki67+, with DNA higher than 2n, and lower or equal to 4n). |
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Comments:
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The panel described in this experiment can be exploited for in depth-analysis of T cell cycle in conditions characterized by altered proportions, numbers and proliferative state of spleen T cell subsets, for example aged mice having higher percentages of memory CD4+ and CD8+ T cells, with or without oligoclonal expansion; lymphopenic mice having compensatory T cell proliferation; or genetically modified mice with abnormal Treg cells representation. Furthermore, this panel may be instrumental in identifying hitherto overlooked changes in Treg and/or naïve/memory T cell subset cycling in a variety of settings such as vaccination, infection, autoimmunity, and cancer.
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