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

## TL;DR

This paper describes methods to measure mitochondrial respiration in immune cells from blood and highlights factors affecting the accuracy of these measurements.

## Contribution

The study provides detailed protocols and insights into the reliable assessment of mitochondrial respiration in PBMCs.

## Key findings

- OXPHOS capacity in PBMCs can be reproducibly measured ex vivo with proper protocols.
- Permeabilization with digitonin requires careful handling to ensure accurate respiratory rate measurements.
- Storage time and temperature significantly influence the results of mitochondrial respiration assays.

## Abstract

Human peripheral blood mononuclear cells (PBMCs) can be easily sampled from healthy individuals and patients. Density gradient isolation from human blood or leukocyte concentrates yields a mononuclear cell population of mainly lymphocytes, monocytes, and natural killer (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, that is, a “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 the limits of sample amount and the impact of storage time and temperature and explored critical aspects of digitonin permeabilization. Furthermore, 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 on OXPHOS capacity in PBMCs can be reproducibly assessed ex vivo, but that great care must be taken during permeabilization to achieve correct measures of respiratory rates.