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 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).

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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Immunology and Infection

459 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

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

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 ...

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 ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

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.&#160;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.&#160;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.&#160;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.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...