Genetic Engineering & Biotechnology News

AUG 2018

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GENengnews.com | Genetic Engineering & Biotechnology News | AUGUST 2018 | 17 An alternative method employs the Agilent Seahorse XF Analyzer, a powerful tool that not only quantifies the immediate metabolic reprogramming of neutrophils but also quantifies the oxygen consumption rate associated with NOX2 enzyme activity and ROS production. This method is not com- promised by secondary ROS production, competing reactions, or probe accessibility— factors that often confound other methods. Neutrophils Rapidly Increase Oxygen Consumption Rate upon Activation Human peripheral blood neutrophils (huPBNs) were isolated from fresh whole blood and plated at 4 × 10 4 cells per well in Cell-Tak™-coated XFe96 cell culture mi- croplates. Changes in the oxygen consump- tion rate (OCR) were measured in response to activation by phorbol myristate acetate (PMA)—a potent activator of the NOX2 en- zyme—in the presence of mitochondrial in- hibitors rotenone and antimycin A (Rot/AA). Addition of mitochondrial inhibitors in the assay before neutrophil activation ensures any oxygen consumption from mi- tochondrial respiration is excluded. Figure 1 shows the rapid rise in OCR observed when neutrophils are stimulated with PMA, reach- ing a peak of OCR within 30 min, and de- clining after 60 min to slowly reach the basal values 90–120 min after stimulation. Activation Kinetics Quantified Using Oxygen Consumption Rate Current methods for quantifying neutro- phil activation are typically end-point mea- surements that fail to provide a dynamic and contextual view of this process. In the next series of experiments, activation data were obtained using the XF analyzer while vary- ing the concentration of the activating com- pound PMA. HuPBNs were plated at 4 × 10 4 per well of XF96 cell culture microplates and activated with the indicated concentrations of PMA in the presence of Rot/AA. The real- time OCR data obtained with varying doses of PMA are shown in Figure 2. Oxidative Burst in Neutrophils Requires Glycolysis What fuels this rapid increase in free radi- cal production? Recent evidence suggests that glycolysis is required to support the acti- vation of neutrophils. Glycolysis is measured in parallel for every XF assay, and as a result, this assay provides a novel view into the re- quirements of this cellular energy pathway during activation. The process of glycolysis results in acidification of the cell culture me- dia, which is measured here as the proton efflux rate (PER). The increase in OCR or oxidative burst after activation with PMA is associated with a simultaneous increase of PER indicative of the dependence of neutro- phils on glycolysis during activation. The specificity and functional relevance of the associated activation with glycolysis to meet the energy demand of oxidative burst was validated using the inhibitor 2-deoxy- d-glucose (2-DG). As shown in Figure 3, 2-DG (or vehicle), Rot/AA, and PMA (or vehicle) were serially administered, and OCR and PER were monitored. The imme- diate increase in OCR with PMA treatment was blocked when glycolysis was inhibited by pretreatment with 2-DG. There is an im- mediate parallel increase in PER with PMA treatment, and this increase in PER is like- wise inhibited with 2-DG. Denouement Neutrophil activation with PMA treat- ment causes an immediate increase in OCR, which can be monitored in real time using XF technology. This is indicative of the as- sembly and activation of NOX2 enzyme pathway and ROS production in activated neutrophils. There is a simultaneous increase in PER indicative of the increase in glycolysis to meet metabolic demands. Due to the non- invasive nature of the XF analyzer, down- stream assays (such as PCR assays, ELISAs, and neutrophil extracellular trap assays) may be performed on the same cells. This applica- tion is a specific, kinetic assay performed on live cells in real time, and it provides tempo- ral resolution of neutrophil activation that is not possible with other assays. For Research Use Only. Not for use in diagnostic procedures. It's Here. The most highly anticipated new journal of 2018 It's Here. Now available in print and online Subscribe at www.crisprjournal.com Brian P. Dranka, Ph.D. (brian.dranka@ agilent.com), is manager of biology, Cell Analysis Division, and Luke Dimasi (luke. dimasi@agilent.com) is manager of global product marketing and technical support, Cell Analysis Division, at Agilent Technologies. Website: www.agilent.com. OMICS

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