Background

Mitochondria play a critical role in maintaining normal cellular function. It is now common knowledge that they not only produce ATP via oxidative phosphorylation but, for example, are also involved in the metabolism of amino acids, lipids and nucleotides, diverse signaling and redox processes as well as quality control and degradation processes including mitophagy and apoptosis (Pfanner et al., 2019). However, mitochondria represent the major site of ATP synthesis in normal cells (Akbari et al., 2019). For this purpose, an electrochemical proton gradient is generated across the mitochondrial inner membrane through the multi-subunit enzyme complexes I–IV. This proton gradient is used by the ATP synthase, also known as complex V, to turn ADP into ATP (Chaban et al., 2014).

The process of oxidative phosphorylation is associated with the reduction of oxygen to water. Accordingly, the oxygen consumption rate of cells can be used for assessing mitochondrial function (Smolina et al., 2017). This principle is the basis of Seahorse XF Analyzers (Agilent Technologies). They provide the possibility to measure not only oxygen consumption rate (OCR), but also the rate of extracellular acidification (ECAR), which is a key indicator of glycolysis. The realtime measurements are carried out in multi-well plates which are provided with solid-state sensors consisting of two fluorophores. One is quenched by oxygen (O₂) and the other one is sensitive to pH-value changes. The fluorophores are excited via light-emitting fiber optic bundles, which subsequently detect the fluorescence changes as a result of oxygen consumption or extracellular acidification (Plitzko and Loesgen, 2018). Furthermore, XF Analyzers enable up to four different injections per well during the measurement. All the properties mentioned constitute a significant advantage over the conventionally used clark-type oxygen electrodes for determining oxygen consumption.

Peripheral blood mononuclear cells (PBMCs) as sample in a XF Analyzer implies that the cells have to be attached to the surface of the microplates. Most commonly, this immobilization is done by means of protein solutions such as Cell-Tak^TM (Jones et al., 2015; Traba et al., 2016; Lee et al., 2019) or Poly-D-Lysine (Hartman et al., 2014; Nicholas et al., 2017; Thaventhiran et al., 2019). We examined both Cell-Tak^TM and Poly-D-Lysine and considered Poly-D-Lysine as most suitable coating method. Since we conducted the Agilent Seahorse XF Cell Mito Stress Test, optimal concentrations of the injected compounds oligomycin, FCCP, antimycin A and rotenone had to be tested as well. A typical curve of a Mito Stress Test is shown in Figure 1. Since oligomycin is an inhibitor of ATP synthase, OCR decreases after its injection. In contrast, OCR increases sharply after FCCP injection, which is an uncoupler of oxidative phosphorylation. The last injection of antimycin A and rotenone, again leads to a decline of OCR, as these two compounds inhibit complex III respectively I of the electron transport chain. The resulting curve is used to calculate various parameters of mitochondrial function (see Figure 1).

Figure 1. Assessment of mitochondrial respiration parameters by means of Agilent Seahorse XF Cell Mito Stress Test. On the left side a typical course of an OCR measurement with injections of oligomycin after the third, FCCP after the sixth and antimycin A together with rotenone after the ninth measuring point is shown. From this curve, various parameters can be calculated, which are marked in different colors. The calculations of the parameters are shown on the right side.

The protocol described in detail hereafter has been applied to examine the influence of 12 days of in vivo caloric reduction in humans. A significant increase of mitochondrial respiratory parameters could be detected in PBMCs of a subgroup of the test persons (Schöller-Mann et al., 2020). Besides such ex vivo studies the protocol can be used to screen any soluble substance, including pharmaceuticals, dietary supplements or contaminants, due to their in vitro effects on PBMCs.