This work was funded by the British Heart Foundation (PG/15/40/31522), Alzheimer Research UK (ARUK-PG2017A-3), and European Research Council (#10102507) grants to G. Pula.

The collection of peripheral blood from consenting volunteers is approved by the local Ethics Committee and the National Health Service Health Research Authority (REC reference: 21/SC/0215; IRAS ID: 283854).
1. Method 1: Superoxide anion detection using DHE by flow cytometry
<ol>
	<li>Pre-warm (37 &#176;C) sodium citrate solution (4% w/v) and use it as an anti-coagulant by adding it directly to the blood at the time of venepuncture at a ratio of 1:7 (0.5% w/v final).</li>
	<...

Detection of Superoxide Anion in Platelets Using Fluorescence Imaging and EPR

In this manuscript, we present three different techniques with the potential to advance the capability to investigate the redox-dependent regulation of platelet function via the selective detection of O2&#8226;-. The first two methods are an improvement on existing techniques because of the redox probe utilized (DHE instead of the more common but less reliable DCFDA). These techniques are, therefore, easily accessible, and most laboratories can adopt them effectively without particular eq...

The superoxide anion (O2&#8226;-) is the most functionally relevant ROS generated in platelets1. O2&#8226;- is the product of the reduction of molecular oxygen and the precursor of many different ROS 2. The dismutation of O2&#8226;- leads to the generation of hydrogen peroxide (H2O2) via spontaneous reactions in aqueous solution or reactions catalyzed by superoxide dismutases (SODs3). Although different enzymatic sources have been suggested (e.g., xanthine oxidase4, lipoxygenase5, cyclooxygenase6, and nitric oxide synthase7), mitochondrial respiration8,9 and nicotinamide adenine dinucleotide phosphate-oxidases (NOXs)10 are the most prominent sources of superoxide anion in eukaryotic cells. This also seems to be the case in platelets, where electron leakage from mitochondrial respiration11,12 and the enzymatic activity of NOXs13,14 have been described as the main contributors to the superoxide anion output.
Although several studies have focused on the regulation of platelets by O2&#8226;-, there is no consensus regarding the molecular mechanisms responsible. The modulation of surface receptor activity via direct oxidation and disulfide bond formation has been proposed for different platelet receptors. The positive regulation of integrin &#945;IIb&#946;3 by ROS via direct oxidation of cysteine residues has been suggested15,16,17. Similarly, since platelet responses to collagen depend on disulfide-dependent dimerization and consequent dimerization of the glycoprotein VI (GPVI)18, receptor activity potentiation by ROS-dependent oxidation has been proposed19, although not fully proven experimentally. Finally, ROS-induced oxidation of the sulfhydryl groups of glycoprotein Ib (GPIb) was shown to promote platelet adhesion and platelet-leukocyte interaction during inflammation20. Conversely, as a possible consequence of decreased sulfhydryl group oxidation and receptor activation, the shedding of the ectodomain of both GPVI and GPIb is diminished&#160;by reducing conditions21.
Modes of action independently of a direct oxidation of platelet surface receptors have also been proposed. ROS, including O2&#8226;-, have been shown to positively modulate the collagen receptor GPVI by attenuating the activity of the Src homology region 2-containing protein tyrosine phosphatase 2 (SHP-2), which negatively regulates the signaling cascade of this receptor22. Moreover, O2&#8226;- can generate ONOO- (peroxynitrite) by rapid reaction with nitric oxide (NO), which normally inhibits platelets through the NO-sensitive guanylyl cyclase (NO-GC) and the generation of the negative platelet regulator cyclic GMP (cGMP)23,24. The resulting decrease in NO levels can lead to platelet potentiation. Alternatively, the generation of O2&#8226;- by NOX2 has been suggested to contribute to lipid peroxidation and isoprostane formation, which is essential for platelet activation and adhesion25. Finally, mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 5 (ERK5), a protein kinase proposed as a redox stress sensor in platelets26, is activated by O2&#8226;- and induces a procoagulant phenotype in platelets (as estimated by flow cytometry-based measurement of phosphatidylserine externalization)27.
The dysregulation of O2&#8226;- and other ROS generation in platelets has been associated with the exaggerated hemostatic response leading to thrombotic cardiovascular complications associated with atherosclerosis, diabetes mellitus, hypertension, obesity, and cancer28,29. In these pathological settings, the ROS output by platelets is increased, which leads to a potentiation of their adhesive and aggregatory responses. In addition to the effect on platelet responses, the free radical output of platelets may have consequences on other blood cells and vascular structures, which is a poorly understood and underinvestigated area of cardiovascular health30. Despite our limited understanding of the molecular mechanisms linking oxidative stress to thrombotic conditions, the clinical relevance of antioxidants for the protection against cardiovascular disease has received considerable attention. Plasma antioxidant levels have been shown to inversely correlate with the risk of developing cardiovascular conditions, and dietary antioxidant consumption has been shown to protect against coronary artery disease31,32.&#160;Consequently, the use of dietary antioxidants has been advocated as a promising approach for cardiovascular disease prevention33,34,35. Amongst the effects of ROS generation in platelets, the increase in apoptosis can have important pathophysiological effects36,37. Overall, reliable protocols to detect and quantify the O2&#8226;- output by platelets are increasingly relevant in cardiovascular research.
Currently, available techniques for the detection of ROS have important limitations of specificity (i.e., the chemical nature of the oxidant molecules detected is unknown) and reliability (i.e., the unwanted interaction with biological molecules and experimental reagents leads to biased non-physiological results)38,39. The most commonly used approach for the detection of ROS in platelets is based on the use of dichlorodihydrofluorescein diacetate (DCFDA), which is converted to dichlorodihydrofluorescein (DCFH) by intracellular esterases and consequently to the highly fluorescent dichlorofluorescein (DCF) by cellular oxidants, including hydroxyl radicals and peroxidase-H2O2 intermediates40,41. Despite its wide use, serious questions have been raised regarding the reliability of this approach for the measurement of intracellular ROS38. The oxidation of DCFH to DCF can be, in fact, induced by transition metal ions (e.g., Fe2+) or heme-containing enzymes (e.g., cytochromes) instead of ROS42. Moreover, DCFDA is converted by cell peroxidases to its semiquinone free radical form (DCF&#8226;-), which is in turn oxidized to DCF by reaction with molecular oxygen (O2) with the release of O2&#8226;-, which leads to the artificial amplification of oxidative responses41,43,44. Therefore, the detection of intracellular ROS by DCFDA is useful for obtaining initial insights but requires cautious consideration and extensive experimental controls38,39.
This study presents three alternative techniques for the detection and measurement of the key regulator of platelet function O2&#8226;-1. The first technique is the detection using DHE and flow cytometry, which offers advantages of reliability and specificity over DCFDA. The second technique proposed here also utilizes DHE, but the detection method is live-platelet fluorescence imaging, which allows the study of the generation of O2&#8226;-&#160;upon platelet signaling with fast kinetics and single-cell resolution. Finally, a protocol based on the use of the hydroxylamine spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) in EPR resonance experiments offers the possibility of quantifying the rate of O2&#8226;- generation by platelets and comparing it in different conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH)</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-02.1-50mg</td>
			<td>Reagent for EPR (spin probe)</td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences&#160;</td>
			<td>NA</td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Merck/Sigma</td>
			<td>A7030</td>
			<td>For &#956;-slide coating</td>
		</tr>
		<tr>
			<td>Bruker E-scan M (Noxyscan)</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>&#160;NOX-E.11-BES&#160;</td>
			<td>EPR spectrometer</td>
		</tr>
		<tr>
			<td>Catalase&#8211;polyethylene glycol (PEG-Cat.)</td>
			<td>Merck/Sigma</td>
			<td>C4963</td>
			<td>Hydrogen peroxide scavenger (specificity control)</td>
		</tr>
		<tr>
			<td>ChronoLog Model 490+4</td>
			<td>Labmedics/Chronolog</td>
			<td>NA</td>
			<td>Aggregometer</td>
		</tr>
		<tr>
			<td>CM radical</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-20.1-100mg&#160;</td>
			<td>Reagent for EPR (calibration control)</td>
		</tr>
		<tr>
			<td>deferoxamine&#160;</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-09.1-100mg&#160;</td>
			<td>Reagent for EPR</td>
		</tr>
		<tr>
			<td>diethyldithiocarbamate (DETC)&#160;</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-10.1-1g&#160;</td>
			<td>Reagent for EPR</td>
		</tr>
		<tr>
			<td>Dihydroethidium</td>
			<td>Thermo Fisher Scientifics</td>
			<td>D11347</td>
			<td>Superoxide anion probe</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Merck/Sigma</td>
			<td>34869</td>
			<td>For stock solution preparation&#160;</td>
		</tr>
		<tr>
			<td>EPR sealing wax plates</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-A.3-VPM</td>
			<td>Consumable for EPR</td>
		</tr>
		<tr>
			<td>EPR-grade water</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-07.7.1-0.5L&#160;</td>
			<td>Reagent for EPR</td>
		</tr>
		<tr>
			<td>Fibrinogen from human plasma</td>
			<td>Merck/Sigma</td>
			<td>F4883</td>
			<td>For &#956;-slide coating</td>
		</tr>
		<tr>
			<td>FITC anti-human CD41 Antibody</td>
			<td>BioLegend</td>
			<td>303704</td>
			<td>Platelet-specific staining for flow cytometry</td>
		</tr>
		<tr>
			<td>Glass cuvettes&#160;</td>
			<td>Labmedics/Chronolog</td>
			<td>P/N 312</td>
			<td>Consumable for incubation in aggregometer</td>
		</tr>
		<tr>
			<td>Horm Collagen</td>
			<td>Labmedics/Chronolog</td>
			<td>P/N 385</td>
			<td>For platelet stimulation</td>
		</tr>
		<tr>
			<td>ImageJ&#160;</td>
			<td>National Institutes of Health (NIH)</td>
			<td>NA</td>
			<td>ImageJ 1.53t (Wayne Rasband)</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Merck/Sigma</td>
			<td>I7378</td>
			<td>For platelet isolation</td>
		</tr>
		<tr>
			<td>Micropipettes DURAN 50&#181;l</td>
			<td>Noxygen Science trasfer and Diagnostics GmbH</td>
			<td>NOX-G.6.1-50&#181;L</td>
			<td>Consumable for EPR</td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrochloride</td>
			<td>Merck/Sigma</td>
			<td>P2658</td>
			<td>For &#956;-slide coating</td>
		</tr>
		<tr>
			<td>Prostaglandin E1 (PGE1)</td>
			<td>Merck/Sigma</td>
			<td>P5515</td>
			<td>For platelet isolation</td>
		</tr>
		<tr>
			<td>Sodium citrate (4% w/v solution)</td>
			<td>Merck/Sigma</td>
			<td>S5770</td>
			<td>For platelet isolation</td>
		</tr>
		<tr>
			<td>Stirring bars (Teflon-coated)</td>
			<td>Labmedics/Chronolog</td>
			<td>P/N 313</td>
			<td>Consumable for incubation in aggregometer</td>
		</tr>
		<tr>
			<td>Superoxide dismutase&#8211;polyethylene glycol (PEG-SOD)</td>
			<td>Merck/Sigma</td>
			<td>S9549</td>
			<td>Superoxide anion scavenger (specificity control)</td>
		</tr>
		<tr>
			<td>Thrombin from human plasma</td>
			<td>Merck/Sigma</td>
			<td>T6884</td>
			<td>For platelet stimulation and &#956;-slide coating</td>
		</tr>
		<tr>
			<td>VAS2870</td>
			<td>Enzo Life Science</td>
			<td>BML-EI395</td>
			<td>NOX inhibitor</td>
		</tr>
		<tr>
			<td>Zeiss 510 LSM confocal microscope</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>Confocal microscope</td>
		</tr>
	</tbody>
</table>

alternative methods for the detection of superoxide anion generation in platelets 

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by&#160;human platelets,&#160;understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually,&#160;identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

Author Spotlight: Innovative Techniques for ROS Detection and Implications for Platelet Research

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets 

The scope of our research is to understand how the generation of reactive oxygen species, regulates platelet function and modulates the hemostatic response. For our work, it is essential to reliably detect the key reactive oxygen species, superoxide anion. The most common technique to detect superoxide anion is flow cytometry with DCFDA as fluorescent probe.These as limitations, it induces the release of reactive oxygen species leading to unreliable data. It does not allow to distinguish, between different reactive oxygen species, and it does not allow to quantify platelet reactive oxygen species. This video presents two alternative techniques for the detection of platelet superoxide anions.The first technique utilizes DHE on live platelets to detect superoxide anion with fast kinetics and single-cell resolution. The second technique utilizes the spin probe, CMH, to quantify the rate of superoxide anion generation by platelets. Overall, the techniques presented in this video, offer significant advantages over most existing techniques, and represent a significant advance in the experimental tools at our disposal to study the redux-dependent regulation of platelets.

For flow cytometry detection of DHE fluorescence, we show representative results for platelets either resting (Figure 3A) or stimulated with 0.1 unit/mL thrombin (Figure 3B). The O2&#8226;- output was quantified as platelet mean fluorescence intensity (MFI), as shown for stimulation with 0.1 unit/mL thrombin (Figure 3C) or 3 &#181;g/mL collagen-related peptide (CRP) (Figure 3D</s...

Read this Scientific Article Protocol about Alternative Methods for the Detection of Superoxide Anion Generation in Platelets at JoVE.com

The generation of superoxide anion is essential for the stimulation of platelets and, if dysregulated, critical for thrombotic diseases. Here, we present three protocols for the selective detection of superoxide anions and the study of redox-dependent platelet regulation.

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ...

alternative-methods-for-detection-superoxide-anion-generation