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>
	<li>Draw peripheral human blood from healthy volunteers by median cubital vein venepuncture.</li>
	<li>Obtain platelet-rich plasma (PRP) from whole blood by an initial centrifugation step at 200 x g for 20 min.</li>
	<li>Centrifuge PRP at 500 x g for 10 min with the reversible platelet inhibitors indomethacin (10 &#181;M) and PGE1 (40 ng/mL).</li>
	<li>Resuspend the resulting platelet pellet in modified Tyrode&#39;s buffer (145 mM NaCl, 2.9 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES),&#160;1 mM MgCl2, 5 mM glucose, pH 7.3) (37 &#176;C) at a density of 2 x 108 platelets/mL.</li>
	<li>After isolation, rest platelets for 30 min at 37 &#176;C.</li>
	<li>Prepare DHE in dimethyl sulfoxide (DMSO) at a stock concentration of 5 mM.</li>
	<li>Add DHE to the platelet suspension at 5 &#181;M final concentration (dilute the stock solution 1 to 1,000, with final DMSO concentration 0.1% v/v) and incubate for 15 min at 37 &#176;C.</li>
	<li>Treat platelets with pharmacological agents and/or ROS scavengers for 15 min before stimulation with the desired physiological stimuli and concentrations (e.g., 0.1 unit/mL thrombin or 3 &#181;g/mL collagen).</li>
	<li>After stimulation, dilute platelet suspensions 1 to 10 in ice-cold modified HEPES buffer.</li>
	<li>For the flow cytometry, set side scattering (SSC) and forward scattering (FSC) gain at 40 mV and 220 mV, respectively, and use logarithmic scale scatter plots to visualize the particle population in the suspension (example in Figure 1A).</li>
	<li>(OPTIONAL) Immunostain one aliquot of platelet suspension using an anti-CD41 antibody to identify the population of events corresponding to platelets (example in Figure 1B).</li>
	<li>Analyse samples by flow cytometry using excitation at 405 nm (violet laser) and emission at 580 nm wavelength, which selectively detects the superoxide anion-specific product 2-hydroxy-ethidium (2OH-Et+) (Figure 2). 
	NOTE: Fixation with paraformaldehyde and long-term sample storage are not advisable.</li>
</ol>
2. Method 2: Superoxide anion detection using DHE by single-platelet fluorescence imaging
<ol>
	<li>Coat an 8-well &#181;-slide on the day of the experiment with 0.1 mg/mL fibrillar collagen I from equine tendons (Horm collagen), 0.2 mg/mL fibrinogen or 1 mg/mL poly-L-lysine (PLL) in modified Tyrode&#39;s buffer for 2 h at 37 &#176;C.</li>
	<li>Wash twice with modified Tyrode&#39;s buffer (10 min each).</li>
	<li>Quench non-specific adhesion by incubation in modified Tyrode&#39;s buffer plus 5 mg/mL bovine serum albumin (BSA) for a minimum of 30 min (or until the experiment).</li>
	<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>
	<li>Draw peripheral human blood from healthy volunteers by median cubital vein venepuncture.</li>
	<li>Obtain platelet-rich plasma (PRP) from whole blood by an initial centrifugation step at 200 x g for 20 min.</li>
	<li>Centrifuge PRP at 500 x g for 10 min with the reversible platelet inhibitors indomethacin (10 &#181;M) and PGE1 (40 ng/mL).</li>
	<li>Resuspend the resulting platelet pellet in modified Tyrode&#39;s&#160;buffer (145 mM NaCl, 2.9 mM KCl, 10 mM HEPES, 1 mM MgCl2, 5 mM glucose, pH 7.3) (37 &#176;C) at a density of 4 x 107 platelets/mL.</li>
	<li>After isolation, rest platelets for 30 min at 37 &#176;C.</li>
	<li>Prepare DHE in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM.</li>
	<li>Add DHE at 10 &#181;M final concentration to the platelet suspension (dilute the stock solution 1 to 1,000, with final DMSO concentration 0.1% v/v) and incubate for 1 min (37 &#176;C).</li>
	<li>After the 1 min of incubation with DHE, remove the blocking solution from the chosen wells, position the &#181;-slide on the microscope stage ready for imaging, and gently dispense platelets.</li>
	<li>Monitor DHE conversion to 2OH-Et+ by inverted confocal imaging for 10 min (405/580 nm ex/em), with images collected every 10 s with a 40x oil immersion lens.</li>
	<li>(OPTIONAL) For wells coated with PLL, monitor the superoxide anion generation in response to a soluble agonist (e.g., 0.1 unit/mL thrombin or 3 &#181;g/mL collagen) by adding the agonist 10 min after platelet dispensing and fluorescence image collection for a further 10 min, as described in step 2.13.</li>
	<li>(OPTIONAL) If required, add superoxide anion scavengers or selective inhibitors (e.g., NOX inhibitor) by dispensing gently to the side of the well during the time course and collect fluorescence images as described in 2.13 for a further 10 min.</li>
	<li>Quantify single-cell fluorescence analysis by selecting the Region of Interest (ROI) containing representative single platelets and analyzing fluorescence intensity in different frames with the Measure tool of imaging suite ImageJ 1.53t.</li>
</ol>
3. Method 3: Detection of superoxide anion by platelets using electron paramagnetic resonance (EPR)
<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>
	<li>Draw peripheral human blood from healthy volunteers by median cubital vein venepuncture.</li>
	<li>Obtain platelet-rich plasma (PRP) from whole blood by an initial centrifugation step at 200 x g for 20 min.</li>
	<li>Centrifuge PRP at 500 x g for 10 min with the reversible platelet inhibitors indomethacin (10 &#181;M) and PGE1 (40 ng/mL).</li>
	<li>Resuspend the resulting platelet pellet in modified Tyrode&#39;s&#160;buffer (145 mM NaCl, 2.9 mM KCl, 10 mM HEPES, 1 mM MgCl2, 5 mM glucose, pH 7.3) (37 &#176;C) at a density of 2 x 108 platelets/mL.</li>
	<li>After isolation, rest platelets for 30 min at 37 &#176;C.</li>
	<li>Add 5 &#181;M diethyldithiocarbamate (DETC) and 25 &#181;M deferoxamine to the platelet suspension.</li>
	<li>Without incubation, add 200 &#181;M 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH).</li>
	<li>Load samples onto the aggregometer within aggregation cuvettes (including Teflon-coated stirring magnets).</li>
	<li>After 1 min, add the stimuli at the desired concentration (e.g., 1 unit/mL thrombin or 3 &#181;g/mL collagen). Incubate for 10 min.</li>
	<li>(OPTIONAL) Obtain superoxide anion generation at different time points by proceeding to step 3.12 at the desired time.</li>
	<li>Transfer the platelet suspension from the aggregation cuvette to a microcentrifuge tube and quickly spin down at 6,000 x g for 10 s.</li>
	<li>Load 50 &#181;L of the supernatant in capillary micropipettes and seal with EPR&#160;sealing wax.</li>
	<li>Transfer samples to the EPR scanner.</li>
	<li>Set the EPR scanner as follows: center field 3,492.5 G, field sweep 60 G, modulation amplitude 2 G, sweep time 10 s, number of scans 10, microwave frequency 9.39 GHz, microwave power 20 mW, conversion time 327.68 ms, time constant 5242.88 ms.</li>
	<li>Estimate CMH oxidation to CM&#9679; as the area under the curve (AUC) of the EPR peaks recorded using the above parameters.</li>
	<li>Build a calibration curve plotting the EPR intensity measured as described in step 3.16 on the y-axis and&#160;the concentrations of the commercially available CM&#9679; on the x-axis (e.g., 0, 0.3, 1, 3, 10, and 30 &#181;M).</li>
	<li>From the CM&#9679; concentration in the samples, obtain the amount of superoxide anion generated by the platelets in the samples using the formulas described in the Representative Results section.</li>
</ol>

Detection of Superoxide Anion in Platelets Using Fluorescence Imaging and EPR

The authors have nothing to disclose.

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><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&amp;nbsp;</td><td>NA</td><td>Flow cytometer</td></tr><tr><td>Bovine Serum Albumin</td><td>Merck/Sigma</td><td>A7030</td><td>For &amp;mu;-slide coating</td></tr><tr><td>Bruker E-scan M (Noxyscan)</td><td>Noxygen Science trasfer and Diagnostics GmbH</td><td>&amp;nbsp;NOX-E.11-BES&amp;nbsp;</td><td>EPR spectrometer</td></tr><tr><td>Catalase&amp;ndash;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&amp;nbsp;</td><td>Reagent for EPR (calibration control)</td></tr><tr><td>deferoxamine&amp;nbsp;</td><td>Noxygen Science trasfer and Diagnostics GmbH</td><td>NOX-09.1-100mg&amp;nbsp;</td><td>Reagent for EPR</td></tr><tr><td>diethyldithiocarbamate (DETC)&amp;nbsp;</td><td>Noxygen Science trasfer and Diagnostics GmbH</td><td>NOX-10.1-1g&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Reagent for EPR</td></tr><tr><td>Fibrinogen from human plasma</td><td>Merck/Sigma</td><td>F4883</td><td>For &amp;mu;-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&amp;nbsp;</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&amp;nbsp;</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&amp;micro;l</td><td>Noxygen Science trasfer and Diagnostics GmbH</td><td>NOX-G.6.1-50&amp;micro;L</td><td>Consumable for EPR</td></tr><tr><td>Poly-L-lysine hydrochloride</td><td>Merck/Sigma</td><td>P2658</td><td>For &amp;mu;-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&amp;ndash;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 &amp;mu;-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).

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

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

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.

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 ...

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

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469 Views.  South Mimms laboratories. 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.

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

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Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

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Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Biology

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Medicine

Analyzing Platelet Phosphatidylserine and Microvesicle Release

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

Isolating Quiescent Platelets for Metabolic Research

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%&#8211;40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.

Platelet metabolism is of interest, particularly as it relates to the role of platelet hyper- and hypoactivity in bleeding and thrombotic disorders. Isolating platelets from plasma is necessary for some metabolic assays; presented here is a method for isolating of intracellular metabolites from washed platelets.

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated ...

The Effects of Shear Stress on Platelets Treated with Antibodies Against Mechanosensory Receptors

Source: Quach, M. E. et al., <a href="https://app.jove.com/v/59704">A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry.</a> J. Vis. Exp.&#160;(2019)
This video demonstrates the method to assess the impact of shear stress on platelets. Antibody treatment induces dissociation and conformational changes in mechanoreceptor complexes, activating the platelets and expressing markers on the surface. These markers are observed via immunostaining and flow cytometry.

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

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Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

Wpływ naprężenia ścinającego na płytki krwi leczone przeciwciałami przeciwko receptorom mechanosensorycznym