2433-P: Comprehensive Assessment of Mitochondria Complex Activity in Mature Adipocytes Using XF-Plasma Membrane Permeabilizer

Introduction and Objective: Mechanistic studies using isolated mitochondria in the XF analyzer provide direct access to electron transport chain (ETC) complexes, precise substrate control, and often clearer interpretation of mitochondrial activity compared to intact cell studies. However, isolating mitochondria from cultured cells, in particular adipocytes, is technically challenging and frequently results in low yield or poor quality. The Agilent XF Plasma Membrane Permeabilizer (PMP), a recombinant cytolysin with selective affinity for cholesterol, permeabilizes the plasma membrane while preserving mitochondrial integrity. This study aims to establish a workflow to assess mitochondria complex activities in permeabilized mature adipocytes. Methods: Using mature 3T3-L1 adipocytes as a model, we established a workflow with an optimized XF-PMP concentration and adjusted the differentiation protocol to prevent oxygen depletion when uncoupled OCR is measured under substrate-replete conditions. To interrogate complex I, II, and IV activities within the same sample, complex-specific substrates and corresponding inhibitors were sequentially injected using the XF analyzer’s automated injection system. Finally, we evaluated the mechanism and potency of metformin-induced mitochondrial inhibition across different concentrations. Results: With optimal dose of XF-PMP, mitochondria function in mature adipocytes remained stable for at least four hours, allowing us to develop a workflow that enables extended assays of ETC complex activity and simultaneous assessment of multiple complexes in the same well. Conclusion: The Agilent XF-PMP reliably permeabilizes mature adipocytes while maintaining mitochondrial health, providing a practical strategy to measure ETC complex activities, screen for mitochondrial modulators, identify gene targets, and characterize drug mechanisms of action. This approach may also support broader applications that require selective plasma membrane permeabilization. Disclosure G. Wang: Employee; Current; Agilent Technologies. L. Winer: None. N. Romero: Employee; Current; Agilent Technologies. Stock/Shareholder; Current; Agilent Technologies.