Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis

Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes. grants: The Hematology Training Program HL007501 (FR), The Inflammatory Disorders Training Grant AI1089673 (DN), The Metabolism, Endocrinology and Obesity Training Grant DK007201 (DN), and the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office www.icb.uscb. edu (DL). TG and ER are paid employees of BioEnergetics LLC, a commercial entity that provided financial support in the form of salaries, research materials, specialized equipment and technical support for assistance in study design, data collection and analysis. The specific roles of these authors are articulated in the author contributions section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.