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

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Protein localization, specifically nuclear versus cytoplasmic, is critical for transcription factor (TF) function in lymphocytes.
• Forkhead box O1 (FOXO1) TF localization regulates gene expression, influencing lymphocyte development, activation, and effector functions.
• Traditional methods like Western blotting lack per-cell resolution and require large cell numbers for protein localization analysis.

Purpose of the Study:

• To develop and optimize an imaging flow cytometry (IFC) protocol for assessing native FOXO1 subcellular localization in human lymphocytes.
• To enable precise quantification of dynamic FOXO1 localization in individual cells and specific lymphocyte subpopulations.

Main Methods:

• Development of a standardized IFC protocol for analyzing native FOXO1 localization.
• Utilized a human CD4+ lymphocyte line (HuT102) and primary human T cells.
• Stimulated cells with PMA/Ionomycin to mimic lymphocyte receptor signaling and induce dynamic FOXO1 translocation.

Main Results:

• IFC successfully quantified dynamic changes in FOXO1 nuclear and cytoplasmic localization on a per-cell basis.
• The protocol allowed for precise analysis within user-defined lymphocyte subpopulations.
• Demonstrated the ability to capture dynamic FOXO1 localization changes lost in bulk analysis methods.

Conclusions:

• IFC provides a powerful tool for studying FOXO1 localization dynamics in human lymphocytes.
• This method facilitates simultaneous analysis of multiple lymphocyte subpopulations and their distinct FOXO1 localization patterns.
• Quantifying FOXO1 localization via IFC can serve as a valuable indicator of immune cell activation and function in research and clinical settings.