Application Note


Quantitative analysis of neutrophil activation using Agilent Seahorse XF technology


N


eutrophils are phagocytic cells that serve as the main anti-microbial defence and initial


immune


response to a site of infection or injury. Upon neutrophil activation, NADPH oxi- dase (NOX2) generates superoxide and reactive oxygen species (ROS) critical for effective anti-microbial immunity and in inflammatory responses. This process results in significant oxygen consumption, com- monly referred to as ‘oxidative burst’. This study demonstrates how Agilent Seahorse XF Technology can be used for detection and quantification of neutrophil activation and oxidative burst in a live-cell, real-time activation assay by measuring oxygen con- sumption rate (OCR) as an early measure of neutrophil activation. Simultaneous gly- colytic flux measurements accurately quan- tified by real-time proton efflux rates (PER) provide insight into the dependence of neu- trophils on glycolysis during activation.


Oxygen consumption is an early measure of neutrophil activation Human peripheral blood neutrophils (huPBN) were plated in an XFe96 cell cul- ture microplate and activated with phorbol 12-myristate 13-acetate (PMA), a potent activator of NOX2 enzyme. Figure 1 shows the rapid rise in OCR observed when neu- trophils are stimulated with PMA, reaching a peak of OCR within 30 minutes, and declining to basal 90-120 minutes after stim- ulation. The profile of the curve demon- strates the dynamics of oxidative burst that


A


300 400 2DG


Veh. or


Rot/ AA


100 200


0 0 50 100 150 200 Time (min)


PMA or


Veh. PMA


2-DG + PMA Vehicle


includes rapid assembly of the NADPH oxi- dase enzyme complex and then disassembly. Neutrophil mitochondria do not consume a significant amount of oxygen, but to ensure that mitochondrial OCR is excluded, the assay was performed in the presence of inhibitors of mitochondrial


function,


rotenone and antimycin A (Rot/AA). The application model described herein can be adapted for various routine studies on neu- trophil activation to investigate innate immune responses to modulators, drug treatments, and micro-environment cues.


Glycolysis drives neutrophil activation Activated neutrophils rely on glycolysis to meet their rapidly-changing cellular ener- getic and metabolic demands. Glucose metabolism is also important to sustain the


100 200 300 400


0 0 or Veh. PMA


Rot/ AA


Human neutrophils (4 x 104/well)


PMA Vehicle


pentose phosphate pathway that generates NADPH, one of the substrates of NOX2 enzyme and necessary for NET release. In addition to real-time OCR measurements, Agilent Seahorse XF technology allows the simultaneous measurement of PER an indi- cator of glycolysis that is not possible with other methods. The specificity and func- tional relevance of the associated activation with glycolysis to meet energy demand of oxidative burst was validated using glycoly- sis inhibitor 2-Deoxy-D-glucose (2-DG). As shown in Figures 2a and 2b, 2-DG (or vehi- cle) Rot/AA and PMA (or vehicle) (black arrows) 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 pre-treatment with 2-DG (Figure 2a). There is a parallel immediate increase in PER with PMA treatment and this increase in PER is likewise inhibited with 2-DG (Figure 2b). Measurement of lactate accu- mulation during the oxidative burst indicat- ed that the majority of PER are produced from glycolysis and that oxidative burst relies on glycolysis (data not shown).


50


100 150 200 250 Time (min)


Figure 1:Oxygen consumption (OCR) during huPBN activation with PMA


B


100 200 300 400 2DG


Veh. or


Rot/ AA


PMA or


Veh. PMA


2-DG + PMA Vehicle


0 0 50 100 150 200 Time (min)


Figures 2a and 2b:Oxygen consumption (OCR; 2a) and proton efflux rate (PER; 2b) during huPBN activation with PMA, with and without 2-DG (glycolysis inhibitor)


54


Conclusions Neutrophil activation with PMA treatment causes an immediate increase in OCR, which can be monitored in real-time using XF tech- nology. This is indicative of the assembly 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 analyser, downstream assays may be performed on the same cells such as PCR, Elisa and NET assays. This application is a specific and kinetic assay per- formed on live cells in real-time providing temporal resolution of neutrophil activation not possible with other assays. This multidi- mensional measure of neutrophil activation may lead to further insight into the effect of modulators such drug treatments, microenvi- ronment and disease progression during innate immune response.


Drug Discovery World Spring 2018


OCR (pmol/min)


PER (pmol/min)


OCR (pmol/min)


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