ASSESSING THE FEASIBILITY OF SIMULTANEOUSLY INVESTIGATING GLYCOLYSIS AND OXIDATIVE PHOSPHORYLATION USING SEAHORSE XF TEST KITS

Abstract

Metabolism is essential for maintaining immune cell proliferation and function. Many enzymatic processes, primarily glycolysis and oxidative phosphorylation (OXPHOS), enable energy production and generate intermediates critical for synthesizing proteins, lipids, and nucleotides. Modern metabolic studies increasingly employ the Seahorse XF analyzer which measures extracellular acidification and oxygen consumption in real time, providing dynamic insights into glycolytic and OXPHOS rates. To assess these parameters, researchers typically use the Glycolytic Rate Assay Kit and Cell Mito Stress Test Kit. While these kits share some components, their simultaneous use for multi-pathway analysis is not standardized, resulting in increased labor, costs and sample requirements, as well as heightened risks of data processing errors. The aim of this study is to evaluate the feasibility of combining these kits to simultaneously measure glycolysis and OXPHOS using the Seahorse XF assay kits. Materials and Methods. Three experimental approaches were tested: 1) Glycolytic Rate Assay; 2) Cell Mito Stress Test; 3) Combined protocol involving sequential addition of inhibitor solutions from both kits. Main results. Our results demonstrate that combining the tests does not affect the baseline measurements of glycolysis and OXPHOS. Other parameters, such as the maximum glycolytic and respiration rates, compensatory glycolysis, and spare respiratory capacity, were also comparable to individual assay results in combined analyses. Conclusions. Thus, combining components from the Glycolytic Rate Assay Kit and the Cell Mito Stress Test Kit enables simultaneous analysis of a wide range of metabolic parameters in immune cells, ranging from baseline glycolysis and OXPHOS values to its peak values, as well as reserve glycolysis and OXPHOS capacities. This approach preserves data quality while reducing the required sample material and minimizing processing errors. The use of a combined protocol creates the opportunity for an in-depth investigations into the metabolic aspects of immune cell activation and proliferation as well as tumor cells biology.