The Multiparametric Analysis of Mitochondrial Dynamics in T Cells from Cryopreserved Peripheral Blood Mononuclear Cells (PBMCs).

The analysis of mitochondrial dynamics within immune cells allows us to understand how fundamental metabolism influences immune cell functions, and how dysregulated immunometabolic processes impact biology and disease pathogenesis. For example, during infections, mitochondrial fission and fusion coincide with effector and memory T-cell differentiation, respectively, resulting in metabolic reprogramming. As frozen cells are generally not optimal for immunometabolic analyses, and given the logistic difficulties of analysis on cells within a few hours of blood collection, we have optimized and validated a simple cryopreservation protocol for peripheral blood mononuclear cells, yielding >95% cellular viability, as well as preserved metabolic and immunologic properties. Combining fluorescent dyes with cell surface antibodies, we demonstrate how to analyze mitochondrial density, membrane potential, and reactive oxygen species production in CD4 and CD8 T cells from cryopreserved clinical samples.