Imaging flow cytometry: A method for examining dynamic native FOXO1 localization in human lymphocytes.

While flow cytometry can reliably assess surface and intracellular marker expression within small cell populations, it does not provide any information on protein localization. Several key transcription factors (TF) downstream of lymphocyte surface receptors are regulated by nuclear versus cytoplasmic localization, and one such TF is Forkhead box O1 (FOXO1). FOXO1 integrates antigen-binding, co-receptor activation and metabolic signals in lymphocytes, leading to proliferation and differentiation. Importantly, the nuclear or cytoplasmic localization of FOXO1 is key for gene expression leading to different lymphocyte phenotypes. In effector lymphocytes (Teff), for example, lymphocyte receptor (TCR) signaling leads to an Akt-dependent phosphorylation of FOXO1. Phosphorylated FOXO1 is excluded from the nucleus, promoting proliferation and effector functions. In contrast, nuclear retention of FOXO1 is essential for early and late development of T and B cells and for the thymic development and stability of regulatory T cells. Given the critical role of FOXO1 localization as an indicator and determinant of function, quantification of FOXO1 cellular localization in human lymphocytes can help determine immune cell activation and activity in experimental and clinical scenarios. The standard method used to determine subcellular protein localization is the analysis of nuclear and cytoplasmic protein extracts by Western blotting (WB). However, available techniques, such as WB, are limited by a requirement for a large number of cells and inability to determine FOXO1 localization in individual cells or sub-populations. In contrast, a standardized method using an imaging flow cytometer (IFC) such as the Amnis Imagestream Mark II, would provide both qualitative, per-cell localization information, as well as quantitative data on gated sub-populations. To this end, we report the development and optimization of an IFC protocol to examine native FOXO1 localization in human lymphocytes. A human CD4+ lymphocyte line, HuT102, as well as primary human T cells, were assessed for dynamic FOXO1 localization after treatment with a lymphocyte receptor signaling mimic (PMA/Ionomycin). IFC nuclear translocation analysis permitted us to precisely quantify the alterations over time in nuclear and cytoplasmic localization of native FOXO1 on a per cell basis, including within specific, user-defined sub-populations of cells. For human lymphocytes, using IFC to assess and quantify dynamic FOXO1 localization allows the user to simultaneously study multiple lymphocyte subpopulations as well as to delineate differing effects of dynamic FOXO1 localization that may be lost when other available methods are used.