Mitochondrial respiration in human peripheral blood mononuclear cells - methodology and influence of permeabilization and storage

Abstract

Human peripheral blood mononuclear cells (PBMCs) are easily sampled in healthy individuals and patients. Density gradient isolation from human blood or leukocyte concentrates yields a mononuclear cell population of mainly lymphocytes, monocytes and NK cells. PBMCs are vital circulating cells of the immune system and rely on oxidative phosphorylation (OXPHOS) for their energy production. OXPHOS capacity can be assessed using oxygraphy in intact and permeabilized PBMCs and has been used to investigate disorders of the immune system, but also, similarly to platelets, employed as a bioenergetic biomarker i.e. "liquid biopsy" of disease conditions unrelated to immune dysregulation. Here, we present some key aspects of mitochondrial respiration in PBMCs isolated from leukocyte concentrates and whole blood using the Oroboros O2k oxygraph. We assessed limits of sample amount, impact of storage time and temperature, as well as explored critical aspects of digitonin permeabilization. Further, we provide respiratory rates and internal ratios from healthy controls using simple and comprehensive protocols for intact and permeabilized PBMCs, respectively. We conclude that detailed information of OXPHOS capacity in PBMCs can reproducibly be assessed ex vivo but that great care must be taken during permeabilization to achieve correct measures of respiratory rates.