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.

Decreased Bioenergetic Health Index in monocytes isolated from the pericardial fluid and blood of post-operative cardiac surgery patients

“…CD14 + monocytes isolated from these patient samples were then subjected to the mitochondrial stress test which was performed within 1 h after plating. Injections of oligomycin (0.5 μg/ml), FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone 1 μM) and antimycin A (10 μM), were used to obtain the mitochondrial function profiles as previously described [17]. Figure 1(A) depicts representative bioenergetic profiles of patient monocytes isolated from the blood and PO-PCF as well as the healthy donor monocyte mitochondrial profile.…”

“…Blood was collected in vacutainers (BD Biosciences) containing 1.5 ml of ACD (acid citrate dextrose) solution (trisodium citrate, 22.0 g/l; citric acid, 8.0 g/l and dextrose 24.5 g/l) as anti-coagulant and processed within 2 h of collection using an established protocol [17]. All isolation procedures were designed to prevent activation of the cells during isolation and were performed at room temperature.…”

“…The cellular bioenergetics of the isolated cells was determined using the XF analyser (Seahorse Bioscience) in combination with the mitochondrial stress test [17]. Real-time, non-invasive measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured and correlated to mitochondrial function and glycolysis respectively.…”

Modification of platelet proteins by 4-hydroxynonenal: Potential Mechanisms for inhibition of aggregation and metabolism

“…Approval for collection and use of platelet concentrates was obtained from the University of Alabama at Birmingham Institutional Review Board (Protocol #X110718014). Platelets were isolated from the concentrates as described previously [20]. In brief, the concentrates were centrifuged at 1500 × g for 10 min and the pelleted platelets were washed with PBS containing prostaglandin I 2 (1 µg/ml) prior to determination of platelet count by turbidimetry [20–22].…”

Evaluation of energy potential of fresh and stored bone marrow cells using a fluorescent potential-sensitive probe

“…Vital cationic probes are used to follow the changes of transmembrane potential which may be registered by microscopy and, potentially, by flow cytometry in different cell populations. Modern experimental protocols with microwell incubation under defined conditions and supplements have been proposed for assessment of bioenergetic changes in cell cultures [7]. In this respect, our general approach well fits current trends in searching bioenergetic parameters of stored cell samples.…”