title: Bioenergetics and the oxidative burst: protocols for the isolation and evaluation of human leukocytes and platelets.
abstract: Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.
authors.affiliation: UAB Mitochondrial Medicine Laboratory, Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Kramer
authors.affiliation: UAB Mitochondrial Medicine Laboratory, Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham.
authors.forename: Balu K
authors.initials: BK
authors.lastname: Chacko
authors.affiliation: UAB Mitochondrial Medicine Laboratory, Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham.
authors.forename: Saranya
authors.initials: S
authors.lastname: Ravi
authors.affiliation: UAB Mitochondrial Medicine Laboratory, Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Johnson
authors.affiliation: UAB Mitochondrial Medicine Laboratory, Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham.
authors.forename: Tanecia
authors.initials: T
authors.lastname: Mitchell
authors.affiliation: UAB Mitochondrial Medicine Laboratory, Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham; darley@uab.edu.
authors.forename: Victor M
authors.initials: VM
authors.lastname: Darley-Usmar
mesh_terms: D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism*; D003584:Cytological Techniques / Q000379:methods*; D004734:Energy Metabolism; D006801:Humans; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D016897:Respiratory Burst / Q000502:physiology*
chemical_list: D017382:Reactive Oxygen Species
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