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.
Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America.
Dequina
D
Nicholas
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States of America.
Elizabeth A
EA
Proctor
Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America.
Forum M
FM
Raval
Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America.
Blanche C
BC
Ip
Department of Medicine, Boston University School of Medicine, Boston, MA, United States of America.
Chloe
C
Habib
BioEnergetics LLC, Boston, MA, United States of America.
Eleni
E
Ritou
BioEnergetics LLC, Boston, MA, United States of America.
Tom N
TN
Grammatopoulos
Department of Medicine, Boston University School of Medicine, Boston, MA, United States of America.
Devin
D
Steenkamp
Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America.|Arthritis Center, Boston University School of Medicine, Boston, MA, United States of America.
Hans
H
Dooms
Department of Medicine, Boston University School of Medicine, Boston, MA, United States of America.
Caroline M
CM
Apovian
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States of America.
Douglas A
DA
Lauffenburger
Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America.|Department of Pathology, Boston University School of Medicine, Boston, MA, United States of America.|Department of Molecular and Cell Biology, Boston University School of Medicine, Boston, MA, United States of America.
Barbara S
http://orcid.org/0000-0001-5647-5740
BS
Nikolajczyk
D000465:Algorithms; D015415:Biomarkers; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000276:immunology / Q000378:metabolism; D004734:Energy Metabolism; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007109:Immunity; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism; D055442:Metabolome; D055432:Metabolomics / Q000379:methods; D008928:Mitochondria / Q000276:immunology / Q000378:metabolism*; D010101:Oxygen Consumption
D015415:Biomarkers