Flow cytometry assessment of mitochondrial indices in CD4+T cells from peripheral blood

Mitochondria play a key role in the vital functions of the cell, i.e., energy production, metabolism, respiration, generation of reactive oxygen species, cell division and death. Impairment of these mitochondrial functions is associated with emergence of various diseases. Their amounts and membrane potential are important indices of the mitochondrial condition. To assess these parameters, various fluorochrome-labeled probes are used, which are detectable by flow cytometry. The opportunity of using fluorescent mitochondrial dyes, together with labeled monoclonal antibodies, opens up new prospects for studying the metabolic parameters in various immune cells. The aim of the present study was to assess the mitochondrial state in CD4⁺T lymphocytes by flow cytometry. To search for the differences in mitochondrial indexes, a group of HIV-infected patients receiving antiretroviral therapy (n = 21) and healthy volunteers (n = 23) were compared. Mononuclear cells isolated from peripheral blood were under the study. Using flow cytometry and commercial mitochondria-selective dyes MitoTracker Green and MitoTracker Orange, we determined, respectively, the mitochondrial mass and membrane charge in the total CD4⁺T lymphocyte pool, as well as in the naive and memory cell subsets. It has been shown that the mitochondrial mass and charge in naive CD4⁺T lymphocytes are lower than in memory cells, both in HIV-infected and uninfected subjects. Moreover, we have established that the HIV-infected patients have an increased mitochondrial mass in total CD4⁺T lymphocyte pool and in their memory cell subset, as compared with healthy donors. That increase, however, was not accompanied by the higher membrane charge. Thus, the analysis of mitochondrial mass and membrane potential using flow cytometry and MitoTracker Green/MitoTracker Orange dyes is relatively easy, fast, and informative for preliminary assessment of the mitochondrial state.