Single-plate Runs of Mito Stress And Glycolysis Stress Tests using Adherent or Non-Adherent Cells.

Real-time metabolic analysis provides label-free measurements of mitochondrial respiration and glycolysis, allowing researchers to link cellular energy metabolism with disease mechanisms and therapeutic responses across diverse fields. When comparing energy metabolism across different mice, other animals, humans, or cells under varying treatments, it is crucial to perform measurements within a single run to ensure accurate and reliable results. Moreover, experimental design must consider the cell type being analyzed, for example, whether they are adherent, like bone marrow-derived macrophages (BMDMs), or non-adherent, like lymphocytes. Therefore, a protocol was developed that enables the simultaneous performance of Mito Stress and Glycolysis Stress tests on the same plate, despite the assays requiring distinct drug injections and analytical programs. Separate handling protocols were optimized for analysis of both highly adherent cells (e.g., BMDMs) and non-adherent "floating" cells (e.g., T cells and other lymphocytes). Here, detailed protocols for conducting these assays efficiently and reproducibly are presented.