Novel methods for simultaneous assessment of CD4 and CD8 T cell mitochondrial potential changes (65.39)

The study of mitochondrial membrane potential (ΔΨm) changes in immune cells is important in understanding immune cell apoptosis, mechanism of diseases, and evaluating the action of compounds and drugs that cause mitochondrial stress. Simultaneous identification of T cells along with ΔΨm changes is challenging due to the broad fluorescence of traditional mitochondrial dyes, such as JC-1, in multiple channels. Here we demonstrate results from a novel approach for the simultaneous evaluation of ΔΨm in CD4 & CD8 T cells using simplified cocktails and a mitochondrial responsive dye followed by microcapillary flow cytometry. PBMC’s were treated with a range of compounds including staurosporine and gambogic acid and evaluated for mitochondrial membrane potential changes and annexin V binding on T cells. Our results demonstrate that with most treatments, a greater % of cells exhibit earlier and more sensitive changes in ΔΨm, than Annexin V binding. Further, some donors treated with staurosporine, a significantly greater % of CD8 T cells demonstrated changes in ΔΨm vs. CD4 T cells. Gambogic acid demonstrated a dramatic reduction of ΔΨm and showed increased apoptosis on both CD4 and CD8 T cells simultaneously. The ability to measure mitochondrial membrane potential changes in CD4 and CD8 T cell simultaneously can allow more sensitive identification of immune cell perturbation and provide deeper insights into the mechanism of immune cell apoptosis in disease and drug discovery.