Аssessing feasibility of simultaneous evaluation of glycolysis and oxidative phosphorylation using Seahorse XF test kits

Metabolism is essential for proliferation and function of immune cells. 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 higher risks of data processing errors. The aim of this study was to evaluate the feasibility of combining these kits in order to measure glycolysis and OXPHOS in simultaneous mode using the Seahorse XF assay kits. 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. Our experiments have shown 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 reserved respiratory capacity, were also comparable to individual assay results in combined analyses. When combining the reagent components from Glycolytic Rate Assay Kit and Cell Mito Stress Test Kit, one may perform 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 provides data quality while reducing the required sample material and minimizing processing errors. The use of a combined protocol creates an opportunity for in-depth studies of metabolic aspects of immune cell activation and proliferation as well as tumor cell biology.