The protocols reported in this manuscript were made possible by the accumulated contributions of previous fellows of Dr. Warren Zapol&#39;s laboratory of Anesthesia Research in Critical Care, Department of Anesthesia at Massachusetts General Hospital. We acknowledge the contribution of Drs. Akito Nakagawa, Francesco Zadek, Emanuele Vassena, Chong Lei, Yasuko Nagasaka, Ester Spagnolli and Emanuele Rezoagli.

L.B. receives salary support from K23 HL128882/NHLBI NIH as principal investigator for his work on hemolysis and nitric oxide. LB receives grants from "Fast Grants for COVID-19 research" at Mercatus Center of George Mason University and from iNO Therapeutics LLC. B.Y. is supported by grants from an NHLBI/#R21HL130956 and DOD/The Geneva Foundation (W81XWH-19-S-CCC1, Log DM190244). B.Y. received patents at MGH on the electric generation of nitric oxide.
L.B. and B.Y. have filed patent application for NO delivery in COVID-19 disease PCT application number: PCT/US2021/036269 filed on June 7, 2021. RWC receives salary support from Unitaid as the principal investigator for technology development aimed at decentralized diagnosis of tuberculosis in children located in low-resource settings.

Due to the high sensitivity, chemiluminescence-based assays for the determination of NO and related compounds have a high risk of NO2- contamination. Each reagent (especially the NO2- blocking solution) and dilutant (including ddH2O) used in the experiment should be tested for its NO2- content to correct for background signal. Nitrite is extremely reactive with a half-life in whole blood around 10 min and rapidly generates NO3-. The time that elapses between blood collection and centrifugation, or NO2- blocking, may therefore cause preanalytical errors and should be minimized15. In an assay using plasma samples, collect blood preferably in heparin tubes and centrifuge at 750 x g for 5 min at 4 &#176;C after thiol group blockade with 40 &#956;L of 200 nM NEM produced by dissolving 250 mg of NEM into 10 mL of phosphate-buffered saline (PBS). The stock solution can be stored at 4 &#176;C. Compounds such as nitroarginine, nitroprusside, and nitroglycerin that inhibit NO synthase (NOS) undergo slow non-quantitative conversion to NO, resulting in a baseline elevation. If the use of NOS inhibitors is part of the investigation, it is advised that blockers that do not contain nitro groups be used43. The inlet pressure in the purge vessel should match the continuous negative pressure made by the CLD for sampling. If the inlet pressure is low, the vacuum distillation of the medium may lead to contaminate gas purging lines and chambers, resulting in signal distortion. Furthermore, small volumes of air may enter the purge vessel and react with the solution to generate excessive NO. On the contrary, excessive pressure caused by a flow rate that is higher than sample withdrawal flow might release NO into the atmosphere leading to an underestimation of the signal15. In addition, both the VCl3 in HCl and the I3- in acetic acid protocol require multiple dilutions during preparation and multiple calculations to yield sample concentration. Every step should be carefully recorded as dilutions and calculations are recognized by many groups as the main source of error44.
Addition of NO2- blocking solution (step 2.1.2) causes red blood cells to lyse, reducing all OxyHb to MetHb. It avoids the rapid reduction of iron-containing proteins (primarily OxyHb) by NO2- with subsequent generation of NO3-. Treatment of samples with this solution upon collection is required to correctly measure NO2- and/or NO3-. In contrast, the solution should not be used when planning the NO consumption by cell-free Hb assay. Aliquots of the NO2- blocking solution should be stored together with the samples and used to remove the portion of signal caused by the solution itself. Finally, the dilution factors should be taken into account for calculating the final concentration of the measured metabolite. In the I3- in acetic acid assay, the NO2- concentration is obtained by subtracting the concentration of the aliquot containing AS without HgCl2 (S-NO and Fe-NO) from that of aliquot containing only PBS (NO2-, S-NO, Fe-NO). To calculate the concentration of S-NO (mercury-labile proteins), the concentration of the aliquot treated with AS with HgCl2 (Fe-NOs or mercury-stable nitroso-proteins) should be subtracted from that of the aliquot treated with AS without HgCl2. Before subtracting, the user should recall multiplying each aliquot&#39;s concentration by 0.8181 (270/330) to take into account the dilution factor used during sample preparations (Figure 3). The same concept applies for S-NO-Hb and Hb-NO measurement, where absolute concentrations are a fraction of measured Hb22. Depending on the type of sample, a precise count of the NO3- concentration may require running both the VCl3 in HCl protocol and the I3- in acetic acid protocol to finally subtract the concentration of NO2- from those of NO3- and NO2-. This is not always necessary in whole blood and plasma samples, for example, where NO3- typically far exceeds NO2-.
It is very important to remind that the CLD will be damaged if the NaOH trap is not in proper place when performing a VCl3 in HCl assay due to the corrosivity of HCl. The 1 M NaOH solution used to fill the NaOH trap can be purchased or prepared either by dissolving 4 g of NaOH salt in 100 mL of ddH2O or by diluting 5 mL of 50% NaOH in 100 mL of ddH2O. When further diluted to 10 &#181;M with ddH2O, the NaOH is very useful to remove protein debris by injection in the purge vessel.
The baseline signal detected by the CLD should be stable and within 0-5 mV. A higher baseline signal could be caused by air pollution (by NO and derivatives), and this can be verified by leaving the inlet in the open air. If a higher voltage is observed only in the purge vessel, while contamination of the tank with inert gas should be considered, it is most often due to the persistence of organic remnants or the presence of NO2- in the tube. Washing the purge vessel multiple times with ddH2O can help minimize the baseline voltage. If unsuccessful, the incubation of the purge vessel with 100 mM NaOH for 24-48 h followed by multiple washes with ddH2O helps eliminate pollutants. In the NO consumption by cell-free Hb assay, the required CLD signal is 70-100 mV for at least 20-30 min. If the signal is not stable (i.e., voltage decreases), an extra dose of 10 &#956;L of DETA NONOate can be added to the purge vessel. This can be repeated as needed. It is essential to remember that both unused powder and already expanded DETA NONOate should be kept at -80 &#176;C. This reagent decays rapidly despite optimal storage and maybe underperforming when not freshly prepared, requiring multiple injections into the purge vessel and leading to a less stable isoelectric signal. More importantly, it should be expanded in PBS with pH 7.4 as this optimizes its half-life (56 h at pH 7.4 at room temperature vs. quasi-instant dissociation at pH of 5.0)45. The syringe used for injection of DETA NONOate should be exclusively dedicated to that action and not used for injection of samples.
If the injection of a sample generates a peak that is higher than the upper point of the calibration curve, especially if it generates a voltage higher than 700 mv, it is advised to dilute the sample (2x or 4x) to minimize error. This also applies to the NO consumption assay if the dip caused by sample injection is deeper than the one caused by the injection of the most concentrated sample used in the calibration curve. The evidence of a peak (or a through) in signal after injection that is less than expected or absent may indicate clogging of the precision syringe used for injection. Due to the small volumes used (10-20 &#956;L/sample), particularly if plasma or urine assays are performed, it may be difficult to distinguish between a filled syringe and the absence of the sample due to clogging of the syringe. It is advised to pay attention to the resistance made by the plunger. After confirmation by inspection of the syringe, removal of the obstruction may be attempted by repeatedly washing the syringe with ddH2O and by rubbing the syringe on a delicate task wipe if the obstruction is externally visible. When in doubt, it is advised to eject the sample volume on a task wipe and go back to wash the syringe with ddH2O and try to repeat sample administration. Foaming is a common complication and forces the experiment to be terminated once the height of the liquid column exceeds that of the reaction column, causing signal instability. Exceeding the antifoaming agent dose indicated in the protocol may lead to an increase in the baseline signal and should be avoided. The number of samples causing the formation of excessive foaming becomes predictable when repeated assays are performed, a phenomenon that should inform how the experiment can be optimally planned. An optional step to decrease foaming is to mix plasma or tissue supernatant with cold methanol (ratio 1:1) and centrifuge for 15 min, 13000 x g at 4 &#176;C. It should be considered that methanol causes proteins to precipitate. Only light S-NOs and Fe-NOs that are not proteic in nature are measured. Thereby, this option cannot be adopted in studies on whole blood or in any other case where there is interest in S-NO and Fe-NO proteins.
More reagent solutions other than VCl3 in HCl and the I3-&#160;in acetic acid have been described and successfully adopted. Ascorbic acid specifically reduces NO2-, does not react with NO3- nor S-NOs, and can be used to reduce the bulk of injected samples and calculations in situations where NO2- is the only molecule of interest. The main pitfall is the limited stability of the reagent, requiring more frequent changes of the reagent solution and, thereby, repeated calibrations46. An assay using carbon monoxide and cuprous chloride (CuCl)-saturated cysteine (3C method) can specifically detect S-NOs with a sensitivity that is comparable with I3-&#160;but still requires treatment with and without HgCl2 to avoid non-specific reactions47. To achieve higher precision in signal analysis, experiments are recorded in the .data format so that offline waveform analysis can be completed with different software other than the bundle program shown in the protocol.
Once the first injection is made, the experiment cannot be stopped. If a pause is needed and/or if any condition is changed (composition of the purge vessel medium, purge gas flow, baseline voltage), the experiment is valid only for the samples injected up to that point. A new standard curve should be performed before measuring more samples. The precision allowed by chemiluminescence-based assays comes with a price. The assays are time-consuming for a number of reasons: 1) automation is not possible as manual sample injection is required in duplicates; 2) most reagents that are used cannot be stocked for long periods; 3) each experiment needs to be started by running standard samples at known concentrations for calibration and cannot be paused unless calibration is repeated; 4) each sample is divided into aliquots containing different mixtures of reagents to block specific reactions in order to finally calculate the concentration of specific metabolites by subtracting others.
This work is focused on the device Zysense NOA 280i, which has a detection threshold of &#60;1 ppb NO in the gas phase (corresponding to up to 1 pM of NO in the liquid phase) and a sampling flow of 10-300 mL/min. Other CLDs are available with a bundle tubing system for the measurement of metabolites in the liquid phase. Ecophysics CLDs have a higher sensitivity threshold, 1 ppb in their most sensitive model but the advantage of not requiring an oxygen tank for ozone generation. On the other hand, their reported sampling flow is 1000 mL/min, which implies a higher consumption of the inert gas tank used for the purge vessel. Other CLD machines are dedicated to exhaled NO analysis for clinical use.
Chemiluminescence is the most sensitive and validated method to specifically assess NO consumption by cell-free Hb in the field of hemolysis. Other techniques are available to measure NO, NO2-, NO3-, S-NOs and mercury-labile nitroso compounds. The direct measurement of NO gas can be obtained either by chemiluminescence or with the use of dedicated electrodes that range from the measurement of the exhaled gas to single-cell electrodes. These measure only NO gas, are less expensive (yet more perishable), require calibration more often, and are less precise compared to a CLD31. Nitrites and NO3- have been historically measured with the Griess reaction, a colorimetric technique that in its original version detects both molecules without the possibility of distinguishing their relative concentration in the sample. Modern variants of the technique involve reverse phase chromatography employing separation of the sample in two columns, allowing two independent reactions with NO3- and NO2-. The sensitivity of those methods is micromolar or slightly less, as compared with 1 nM with CLD. The main pitfalls are time consumption and incompatibility with samples pretreated with mercury and/or ferricyanide14. S-NOs can be detected with colorimetric and fluorometric techniques as well with a sensitivity up to 0.5 &#181;M compared to 100 fM described by using chemiluminescence as shown in the I3- in acetic acid protocol14,41,42. The biotin-switch assay, despite failing to detect S-NOs in the picomolar range, has the advantage of generating biotin-labeled proteins that can be purified and identified with proteomic analysis48. When looked at in detail, each technique shares one common pitfall, which is the extreme reactivity of NO and its derivatives mandating blockade of reactions for the purpose of in vitro measurement. Pre-treatment of samples is critical, and answering the question of whether a certain pre-treatment completely blocks a reaction or does so better than another one has been so far a driving force towards an increase in methodological studies. While the extreme reactivity in vivo of NO has certainly been a drive to research sensitive methods for detection of its metabolites, direct measurement of NO has met significant progress in the past three decades since the first amperometric detector was developed in 1990. More than twenty different probes have been described since then, including nanosensors used to study single-cell NO content49. Electrochemical sensors are the main tools that can detect NO both in vivo and in real-time. In pulmonary medicine, multiple studies have compared electrode-based machines with CLDs. Measurement of exhaled NO is more simple than other assays for metabolite detection, requiring either direct connection of the gas line (via IFD filter) to a closed-system respiratory adjunct or a few breaths in a bag for offline analysis, yet the cost and poorer portability of CLDs standstill. A number of electrochemical devices currently meet the criteria for clinical use, although they have lower sensitivity and reproducibility when compared to CLDs. Furthermore, the concordance in terms of absolute measured values is suboptimal between devices, so it is recommended that patients should be always followed up with the same electrochemical device50. The use of CLDs in settings such as measurement nasal exhaled NO and in research settings like therapeutic low and high-dose inhaled NO is still necessary.
Precise measurements of NO, its derivatives, and NO-binding proteins are necessary at many levels to understand an overlooked and still mostly unknown mechanism of signaling. The fields of application span biology and pathology at both preclinical and clinical levels with specific target areas, including immunobiology, neurosciences, cardiovascular sciences, and infective diseases. Preclinical studies and clinical trials are investigating the use of inhaled NO gas as a therapeutic agent. Deeper knowledge on how exogenous NO gas influences the concentration of NO metabolites and NO-bound proteins at every level, from plasma to cell compartments, will be required. Implementation of high-yield proteomics may likely increase the number of known mediators that are influenced by NO and its derivatives and may mandate new mechanistic investigations requiring precise measurements of NO metabolites.

Since Salvador Moncada and Nobel laureates Robert Furchgott, Louis Ignarro, and Ferid Murad identified nitric oxide (NO) as the previously known endothelial-derived relaxation factor (EDRF), the central role of NO has been established in several key mechanisms spanning throughout vascular biology, neurosciences, metabolism, and host response1,2,3,4,5,6,7. Exogenous administration of NO gas has become an established treatment for respiratory failure due to pulmonary hypertension in the newborn8. Nitric oxide gas has also been investigated for treatment of respiratory syncytial virus (RSV) infection, malaria and other infective diseases, ischemia-reperfusion injury, and for prevention of acute kidney injury in patients undergoing cardiac surgery9,10,11,12. The need for precise measurement techniques to assess the levels of NO, its metabolites, and those of its target proteins and compounds arises from both mechanistic and interventional studies.
Due to its high reactivity, NO may undergo different reactions depending on the biological matrix in which it is produced and/or released. In the absence of hemoglobin (Hb) or other oxy-hemoproteins, NO is oxidized almost completely to nitrite (NO2-).
2NO + O2 &#8594; 2NO2
NO2 + NO&#160;&#8594; N2O3
N2O3 + H2O&#160;&#8594; NO2-&#160;+ H+
NO first undergoes autoxidation with molecular oxygen (O2) to yield nitrogen dioxide (NO2) and reacts with NO2 itself to generate dinitrogen trioxide (N2O3). One molecule of N2O3 reacts with water (H2O) to form two molecules of NO2- and a proton (H+)13. Within whole blood14,15, NO and NO2- are rapidly converted to nitrate (NO3-) as these molecules react avidly with the oxidized heme groups of Hb [Hb-Fe2+-O2 or oxyhemoglobin (oxyHb)] to yield NO3-. This reaction is coupled with the transition of the heme group to the ferric state [Hb-Fe3+ or methemoglobin (metHb)]:
Hb-Fe2+-O2 + NO&#160;&#8594; Hb-Fe3+ + NO3-
The red blood cell (RBC) barrier and the space immediately adjacent to the endothelium are the main factors limiting this reaction and allowing a small portion of the NO released by the endothelium to act as EDRF16,17. In fact, cell-free Hb in the circulation is known to disrupt vasodilation in experimental and clinical settings17,18. Within the RBC, depending on oxygenation and NO2- concentration, a portion of NO reacts with deoxyhemoglobin (Hb-Fe2+) to form iron-nitrosyl Hb (Hb-Fe2+-NO or HbNO):
Hb-Fe2+ + NO&#160;&#8594; Hb-Fe2+-NO
In the RBC15,17, NO2- can form Hb-Fe3+ by reducing Hb-Fe2+ leading to the release of NO, which in turn binds Hb-Fe2+-O2 (preferentially) or Hb-Fe2+.
The generation of NO-derivatives should not be considered strictly unidirectional as NO can be regenerated from NO2-&#160;and NO3- in various tissues and by different enzymes (e.g., by intestinal bacteria or within mitochondria, particularly under hypoxic conditions)19,20.
A variable amount of NO produced (or administered) leads to the downstream generation of S-nitrosothiols, mainly by thiol transnitrosation from N2O3 in the presence of a nucleophile creating a NO+ donor intermediate (Nuc-NO+-NO2-):
N2O3 + RS- &#8594; RS-NO + NO2-
Another possibility for S-nitrosothiols generation is nitrosylation of oxidized thiols (NO reacting with an oxidized thiol):
RS&#8226; + NO&#160;&#8594; RS-NO
This mechanism and direct thiol oxidation by NO2 might be possible only in very specific conditions which are described elsewhere21. S-nitrosothiols range from light molecules like S-nitrosoglutathione to large thiol-containing proteins. S-nitrosohemoglobin (S-NO-Hb) is formed by nitrosation of a thiol group of a conserved cysteine residue in the &#946;-chain (&#946;93C)22.
The generation and metabolism of S-nitrosothiols are part of important regulatory mechanisms. Examples include regulation of glutathione, caspases, N-methyl-D-Aspartate (NMDA), and ryanodine receptors23,24,25,26,27,28. Previously considered as a major mediator of NO biology in vivo, nitrosated albumin (S-nitroso-albumin) seems to be a NO/NO+ transporter without any specific additional biological activity29.
When measuring the concentration of NO and its derivatives from a specific biological sample within a biological matrix, it is important to consider characteristics such as acidity, oxygenation, temperature, and the presence of reagents. Examples include administered exogenous NO donors and, in the setting of acute inflammation, hydrogen peroxide (H2O2) reacting with NO2 leading to generation of supernormal concentration of free radicals like peroxynitrite (ONOO-)21. In addition to the analytical method that is employed, the preanalytical phase of sample preparation and storage is fundamental. Downstream reactions that do not represent the in vivo NO activity shall be predicted, considered, and blocked. A valid example is the instability of S-NO-Hb, requiring a dedicated treatment of blood samples when it is targeted for measurement22.
Chemiluminescence-based assays are the gold standard for detecting the levels of NO and its main metabolites [NO2-, NO3-, S-NO and iron-nitrosyl complexes (Fe-NO)] in any biological fluid, including tissue homogenates30,31. These methods rely upon the chemiluminescence detector (CLD), a device that houses the reaction of NO with ozone (O3), generating NO2 in an excited state (NO2&#8226;). Relaxation of NO2&#8226; causes emission of a photon of light that is detected by a photomultiplier tube, generating an electric signal that is directly proportional to the NO content of the sampled gas mixture32. A simplified schematic of the CLD is represented.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63107/63107fig01.jpg" /> 
Figure 1: Simplified schematic of a chemiluminescence detector for nitric oxide gas. Chemiluminescence-based detection of nitric oxide (NO) is the stoichiometric generation of one photon per NO gas molecule that is introduced in the chemiluminescence detector (CLD). The chemiluminescence reaction is obtained in a designated chamber supplied with ozone (O3) from an internal generator, which is kept at negative pressure by connection with an external pump, allowing continuous and constant inflow of sample gas. The generation of O3 requires diatomic oxygen (O2) that is supplied by a dedicated O2 tank connected with the CLD (other manufacturers provide CLDs operating with ambient air). Within the reaction chamber, each molecule of NO gas contained in the sampled gas reacts with oxygen to yield one molecule of nitrogen dioxide in the activated state (NO2*). By returning to its ground state, each NO2* molecule emits one photon that is detected by a photomultiplier tube (PMT) located adjacent to the reaction chamber. The PMT with the associated amplifier and central processing unit produces a signal proportional to the photon count and the number of NO molecules in the reaction chamber. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The sample inlet of the CLD can be connected to a glassware system containing a reaction chamber for liquid samples. The system is continuously purged with an inert gas such as nitrogen, helium, or argon, transferring NO from the reaction chamber to the CLD. Liquid-phase samples are injected through a dedicated membrane into the purge vessel.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63107/63107fig02.jpg" /> 
Figure 2: Structure of a purge vessel for chemiluminescence-based detection of nitric oxide gas&#160;The purge vessel (right) allows for the detection of nitric oxide (NO) gas or any other compound that can be readily converted to NO gas when released from a liquid phase reagent. The inert gas inlet is connected to a source (tank) of an inert gas such as Argon, Xeon, or diatomic nitrogen (N2). The needle valve (opens to the left) is used for pressure control within the purge vessel and can be completely removed to clean the vessel. The injection port is covered by a cap with a membrane septum for sample injection. The membrane should be replaced often. A heated jacket surrounds the reaction chamber and is connected to a hot water bath to perform the VCl3 in HCl assay. The purge vessel outlet is connected to the chemiluminescence detector (CLD) or to the NaOH trap (required for VCl3 in HCl assays). To drain the reaction chamber content, first close the stopcocks at the inert gas inlet and the purge vessel outlet, close the needle valve, remove the cap at the injection port and finally open the stopcock at the drain. The NaOH trap (left) is required to be placed inline between the purge vessel and the CLD if the VCl3 in HCl assay is performed because of the corrosivity of HCl. The connection to the CLD always requires an intense field dielectric (IFD) filter to be placed between the CLD and the output of the purge vessel (or the NaOH trap, if used). The IFD filter removes airborne particles and stops liquid from passing through the purge vessel. PTFE = polytetrafluoroethylene. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As a consequence, any compound that can be converted to NO through a specific and controlled chemical reaction can be detected with high sensitivity in any biological fluid and tissue homogenate24. Direct measurement of gas-phase NO through chemiluminescence is performed in both experimental and clinical settings. These techniques are extensively described elsewhere33,34,35. Measurement of NO2-, S-nitrosothiols, S-nitrosated proteins, and Fe-NOs can be performed by adding samples in a reaction mixture with triiodide (I3-), which stoichiometrically releases NO gas from all these compounds:
I3- &#8594; I2 + I-
2NO2&#8722; +2I&#8722; +4H+ &#8594; 2NO + I2 +2H2O
I3&#8722; + 2RS-NO &#8594; 3I&#8722; + RSSR + 2NO+
2NO+ + 2I&#8722; &#8594; 2NO + I2
while I3- does not react with NO3-15. Precise measurements of each compound are made possible by pre-treatment of sample aliquots with acidified sulfanilamide (AS) with or without mercuric chloride (HgCl2). Specifically, pre-treatment with AS removes all NO2- content. As a consequence, the NO content measured by the CLD only reflects the sum of S-NOs and Fe-NOs concentration. Injection of HgCl2 in a sample aliquot before AS injection causes NO2- to be released by S-NO. Treatment with AS (leading to NO2- removal) ensures that the measured NO content only reflects the concentration of Fe-NOs. A series of subtractions between the assessments allow to calculate the precise concentration of the three NO derivatives22.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63107/63107fig03.jpg" /> 
Figure 3: Steps in sample preparation for the I3- in acetic acid chemiluminescence assay. The sequential steps for preparation of the I3- in acetic acid chemiluminescence assay are illustrated. Use of light-protected centrifuge tubes is required. Tubes 1, 2, and 3 are those used to prepare for the assay. Another sample aliquot (tube 4) is needed for the VCl3 in HCl assay if the measurement of nitrate (NO3-) is required. Steps are indicated by numbers in red. Prefill (Step 1) as indicated with phosphate buffer saline (PBS) or HgCl2 before adding the sample volume. Add the sample volume (2) as indicated, vortex, and incubate for 2 min at room temperature (RT). Add (3) PBS or acidified sulfanilamide (AS) as indicated,vortex, and incubate for 3 min at RT. Run the assay (4). The concentration measured by the assay is the sum of the concentration of the compounds reported under each tube. Tube number 1 will allow measurements of nitrite (NO2-), S-nitrosothiols (S-NO), and iron-nitrosyl complexes (Fe-NOs) as a single signal. For nitrate (NO3-) measurement, samples shall be run with both I3- in acetic acid and VCl3 in HCl assays, and the value obtained from tube 1 should be subtracted from the one obtained from tube 4. *suggested quantities to be used for Hb analysis for determination of residual NO2-, S-nitrosohemoglobin and iron-nitrosyl-hemoglobin. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For NO3- measurement, Vanadium (III) chloride (VCl3) in hydrochloric acid (HCl) is used for conversion of NO3- to NO in the purge vessel in order to measure NO3- stoichiometrically with the CLD:
2 NO3-+ 3V+3 + 2H2O&#160;&#8594; 2NO + 3VO2+ + 4H+
To achieve a sufficiently fast conversion, the reaction needs to be performed at 90-95 &#176;C. Reduction from NO3- to NO2- is coupled with reduction of NO2- to NO by HCl. Vanadium metal also reduces S-NOs liberating their NO moiety22,36. The final concentration obtained by CLD with VCl3 in HCl reflects the aggregate concentration of NO3-, NO2, and other nitrosated compounds. Subtraction of the latter value from the concentration yielded with CLD with I3- allows for the calculation of NO3- concentration36,37 (Figure 3).
In the NO consumption assay, the continuous release of NO in the purge vessel by NO donors like (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) generates a stable signal allowing quantifying cell-free oxyHb in the injected samples. The amount of NO consumed in the purge vessel is in a stoichiometric relationship with the amount of oxyHb in the sample38.
Protocols for measurement of NO2-, NO3-, S-nitrosothiols, iron-nitrosyl complexes, and NO consumption by cell-free Hb in plasma samples are illustrated. Studies on NO in the RBC environment require specific sample treatment followed by exclusion chromatography to measure extremely fragile S-NO-Hb and Hb-NO coupled with the determination of total Hb concentration15,22. Sample preparation is instrumental in correcting measurement. Pre-existence of NO2- in H2O and release of NO2- during the assay can lead to measurement of artificially higher concentrations of NO derivatives such as S-NO-Hb14,39. Important aspects of sample preparation are also presented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma</td>
			<td>45754</td>
			<td>500 mL - liquid</td>
		</tr>
		<tr>
			<td>Antifoam B Emulsion</td>
			<td>Sigma</td>
			<td>A5757</td>
			<td>250 mL - liquid</td>
		</tr>
		<tr>
			<td>DETA NONOate</td>
			<td>Cayman</td>
			<td>82120</td>
			<td>10 mg</td>
		</tr>
		<tr>
			<td>Gibco DPBS (1x) no calcium, no magnesium</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Hydroochloric Acid 37% (1 M)</td>
			<td>Sigma</td>
			<td>258148</td>
			<td>500 mL - liquid</td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>SAFC</td>
			<td>207772</td>
			<td>100 g - solid</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Mercury (II) Chloride ACS reagent&#62; 99.5%</td>
			<td>Sigma</td>
			<td>215465</td>
			<td>100 g - solid (dissolve in water)</td>
		</tr>
		<tr>
			<td>Mili-Q Water Purification System</td>
			<td>Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Model 705 RN 50 &#956;L syringe</td>
			<td>Hamilton</td>
			<td>80530</td>
			<td>Microliter syringe</td>
		</tr>
		<tr>
			<td>Model 802 N 25 &#956;L Syringe</td>
			<td>Hamilton</td>
			<td>84854</td>
			<td>Microliter syringe</td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>4260</td>
			<td>25 g - crystalline</td>
		</tr>
		<tr>
			<td>Nitric Oxide Analyzer + Bundle Software - Purge Vessel</td>
			<td>Zysense</td>
			<td>NOA 280i</td>
			<td>Chemiluminescence Detector</td>
		</tr>
		<tr>
			<td>Nonidet p-40&#160; (NP-40)</td>
			<td>ThermoFisher</td>
			<td>85125</td>
			<td>10% - 500 mL</td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate (III) ACS reagent&#8805; 99%</td>
			<td>Sigma</td>
			<td>244023</td>
			<td>100 g - powder</td>
		</tr>
		<tr>
			<td>Potassium Iodide ACS reagent&#62; 99%</td>
			<td>Sigma</td>
			<td>221945</td>
			<td>100 g - solid</td>
		</tr>
		<tr>
			<td>Potassium Nitrite cryst. For analysis EMSURE ACS</td>
			<td>Supelco</td>
			<td>105067</td>
			<td>250 g - crystalline</td>
		</tr>
		<tr>
			<td>PowerGen 125</td>
			<td>Fisher Scientific</td>
			<td>14-359-251</td>
			<td>Mechanic Homogenizer</td>
		</tr>
		<tr>
			<td>RV3 Two Stage Rotary Vane Pump</td>
			<td>Edwards</td>
			<td>A65201906</td>
			<td>Vacuum Pump - Bundled with analyzer</td>
		</tr>
		<tr>
			<td>Sodium Heparin - BD Hemogard Clo</td>
			<td>BD Biosciences</td>
			<td>BD367871</td>
			<td>75 USP Units</td>
		</tr>
		<tr>
			<td>Sodium hydroxide anhydrous ACS reagent &#8805; 97%</td>
			<td>Sigma</td>
			<td>795429</td>
			<td>1 kg - pelletts</td>
		</tr>
		<tr>
			<td>Sodium Nitrate ACS reagent &#8805; 99%</td>
			<td>Sigma</td>
			<td>221341</td>
			<td>500 g - powder</td>
		</tr>
		<tr>
			<td>Sodium nitrite &#8805; 99%</td>
			<td>Sigma</td>
			<td>S2252</td>
			<td>500 g - crystalline</td>
		</tr>
		<tr>
			<td>Sulfanilamide &#8805; 98%</td>
			<td>Sigma</td>
			<td>S9251</td>
			<td>100 g - solid</td>
		</tr>
		<tr>
			<td>Vanadium (III) Chloride</td>
			<td>Sigma</td>
			<td>112393</td>
			<td>25 g - solid - Caution (exothermic)</td>
		</tr>
		<tr>
			<td>Whatman 1 Filter Paper</td>
			<td>Sigma</td>
			<td>WHA10010155</td>
			<td></td>
		</tr>
	</tbody>
</table>

chemiluminescence-based assays for detection of nitric oxide and its derivatives from autoxidation and nitrosated compounds

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

The NO-consumption by cell-free Hb assay was used in samples containing known concentrations of cell-free oxyHb (Figure 4). As one heme of oxyHb stoichiometrically releases one NO molecule in the assay, purified cell-free oxyHb is used to build the calibration curve for the assay (Supplemental Figure 3).
The dose-response relationship between cell-free Hb (measured with a colorimetric assay) and NO consumption in patients coming off cardiopulmonary bypass (CPB) during cardiac surgery is shown in Figure 5. It can be assumed that after CPB there is a higher concentration of heme groups in the Hb-Fe2+-O2 status (Figure 5, red) compared to patients receiving NO during CPB from which only a minority of heme groups of cell-free Hb consumes NO (Figure 5, green).
The protocol for assessing NO consumption by cell-free Hb was previously applied to test plasma samples from 50 patients undergoing cardiac surgery and requiring CPB. Blood was collected at baseline and 15 min, 4 h, and 12 h after CPB. The purpose of the study was to investigate the existing relationship between both pulmonary and systemic vascular resistance and the increase in hemolysis observed after the CPB. Cell-free Hb concentration was assessed with a Hb colorimetric assay. Hemolysis peaked 15 min after CPB. The accumulated free Hb was slowly eliminated from plasma within 12 h18 (Figure 6A). NO consumption, instead, peaked at 15 min and reached baseline values in just 4 h (Figure 6B). Linear regression curves of NO consumption by cell-free Hb exhibited a stoichiometric relationship at the 15 min post-CPB timepoint as opposed to baseline, 4 h, and 12 h post-CPB (Figure 6C). The explanation for the observed difference in kinetics lies in the abundance of newly released free Hb that has not yet encountered NO molecules being released by the patient&#39;s endothelium. The concentration of oxyHb (scavenging NO and resulting in consumption in the assay) was, in fact, higher at 15 min than at any other timepoint. Plasma collected at baseline and on other timepoints had a relatively higher component of metHb, which had already been oxidized by NO, did not bind NO, and did not cause consumption in the assay. Pulmonary and systemic vascular resistances were measured invasively and peaked at 15 min. For the first time in this specific patient population, NO consumption by free Hb was observed to be temporally coupled with vasoconstriction, which confirmed previous observations in animal models and in patients with sickle cell disease18.
The administration of NO gas during CPB and after surgery increases the relative quantity of circulating metHb vs. oxyHb. When patients undergoing cardiac surgery were treated with NO-gas intra-operatively, and post-operatively, the observed increase of free Hb after CPB (Figure 7A) was not coupled with any increase in NO consumption, as measured with the aforementioned protocol (Figure 7B). The exogenously administered NO gas converts most oxyHb into metHb, which in turn does not scavenge NO in vivo nor consumes NO in vitro (unpublished data).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63107/63107fig04.jpg" /> 
Figure 4: Nitric oxide consumption by purified cell-free oxyhemoglobin. Nitric oxide consumption assay performed by injecting samples of known concentrations of purified cell-free oxyhemoglobin. Regression line is shown in red. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63107/63107fig05.jpg" /> 
Figure 5. Nitric oxide consumption and cell-free hemoglobin concentration from patients undergoing cardiac surgery with cardiopulmonary bypass. Each patient&#39;s cell-free hemoglobin level, as measured with a colorimetric kit, was plotted with the nitric oxide (NO) consumption measured through chemiluminescence. Blood samples were drawn 15 min after the end of CPB. Regression lines are shown for cardiac surgery patients not receiving (in red) or receiving (in green) NO during cardiopulmonary bypass (CPB). Hemoglobin is expressed in &#956;M of heme groups. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63107/63107fig06.jpg" /> 
Figure 6: Nitric oxide consumption and cell-free hemoglobin in cardiac surgery patients. (A) Cell-free hemoglobin (Hb) concentration in the plasma of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) (N = 50) was determined at different time points with the use of a commercially available colorimetric determination kit. Data were analyzed by fitting a mixed model with evidence of a fixed effect between time points. Plasma concentration at different time points was compared to baseline with Tukey&#39;s multiple comparison test. (B) Nitric oxide (NO) consumption by plasma obtained from the same samples used for quantification of free Hb. Data were analyzed by fitting a mixed model with evidence of a fixed effect between time points. Plasma concentration at different time points was compared to baseline with Dunnett&#39;s multiple comparison test. (C) Regression lines for cell-free Hb plasma content matched with NO consumption. * p &#60; 0.05, ** p &#60; 0.01, *** p &#60; 0.001, **** p &#60; 0.0001, ns not significant. Data are presented as median &#177; interquartile range. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63107/63107fig07.jpg" /> 
Figure 7: Effects of nitric oxide gas administration on nitric oxide consumption in patients undergoing cardiac surgery. (A) Cell-free hemoglobin concentration in the plasma of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) either receiving a placebo (N = 28) or nitric oxide (NO) gas (N = 22) for 24 h since the beginning of CPB. The concentration was determined at different time points with the use of a commercially available colorimetric kit. Data were analyzed by Friedman&#39;s test, indicating the presence of an effect between time points in both groups. Plasma concentration at different time points was compared to baseline with Dunn&#39;s multiple comparison test. (B) NO consumption by plasma obtained from the same samples used for quantification of cell-free Hb. Data were analyzed by Friedman&#39;s test, indicating the presence of an effect between time points in both groups. Plasma concentration at different time points was compared to baseline with Dunn&#39;s multiple comparison test. ** p &#60; 0.01, *** p &#60; 0.001. Data are presented as median &#177; interquartile range. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Assay</td>
			<td>Reagent Mixture</td>
			<td>Target Baseline (mV)</td>
		</tr>
		<tr>
			<td>VCl3 in HCl</td>
			<td>5 mL of VCl3 in HCl saturated solution + 100 &#956;L of antifoam agent</td>
			<td>0&#8211;5 mV*</td>
		</tr>
		<tr>
			<td>I3&#8211; in acetic acid</td>
			<td>5&#8211;7 mL of I3&#8211; reagent</td>
			<td>0&#8211;5 mV*</td>
		</tr>
		<tr>
			<td>NO consumption by cell-free Hemoglobin</td>
			<td>5&#8211;7 mL of PBS at pH 7.4 + 100 &#956;L of antifoam agent + 20 &#956;L of expanded DETA-NONOate solution</td>
			<td>70&#8211;100 mV**</td>
		</tr>
		<tr>
			<td colspan="3">*ideal value: baseline might be higher due to the quality of the air in the experiment environment</td>
		</tr>
		<tr>
			<td colspan="3">**ensure stability for 20 min before starting the experiment.</td>
		</tr>
	</tbody>
</table>
Table 1: Purge vessel reagent composition and target baseline for various chemiluminescence assays. Purge vessel reagent composition for each described assay. NO = Nitric Oxide; PBS = Phosphate Buffer Saline; DETA-NONOate: (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. *ideal value: baseline might be higher due to the quality of the air in the experiment environment. **ensure stability for 20 min before starting the experiment. To perform the NO consumption assay, the scale should be changed on the software to capture values between 70 mV and 100 mV (e.g., 0-100 mV), which is the targeted baseline voltage. This adjustment is suggested to have full control on the sample injection, which enables to document the signal moving from baseline following the injection of a sample and to observe the signal returning to baseline. Data recording is not influenced by nor limited to the visualized scale.
Supplemental Figure 1: Peaks following NO detection in the VCl3 in HCl assay. The peaks which follow sample administration in the VCl3 in HCl are shown on the CLD bundle software. Calculation of the area under the peak for each injection is shown for convenience and explained in Supplemental Figure 2. In this experiment, standards of different concentrations have been administered for the calibration curve, followed by plasma samples. Dilution is a useful solution to calculate metabolites from samples that yield NO at the high end or even outside of the calibration curve. <a href="https://www.jove.com/files/ftp_upload/63107/Supplemental Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure 2: User-assisted automated calculation of the area under the peak. When performing the NO consumption assay by cell-free hemoglobin, it may be necessary to set peaks manually in order to provide the software with the most precise limits to measure the area under the peak generated by each sample injection. (A) Representative full view of a recorded experiment. It is reached by clicking on Process in the main menu and by selecting and opening the recorded file of interest. The yellow line represents the signal threshold (mV) that constitutes the straight segment of the area under the peak. By clicking on it, the user can drag it towards the signal area. The x and y-axis can be changed to zoom on the peak of interest for precise measurement by either clicking on the lower left buttons or by typing in the desired minimum and maximum value for each. (B) Threshold positioning. The user can select the optimal baseline threshold by dragging the yellow line to the signal midpoint right prior to the signal downslide caused by the sample injection. To continue limiting the area, the user shall click on the Set Peaks Manually button. (C) Start and End Cursors positioning. By clicking on the Set Start Cursor button, a green line appears on the screen. The green line should be placed (by dragging and releasing) at the very beginning of the downslide caused by the sample injection. The same button changes its label to Set End Cursor by clicking on which a red line appears on the screen. The red line should be placed at the point where the signal reaches the baseline again after the deflection caused by NO consumption from the injected sample. Altogether, the threshold line (yellow, B), the green line, and the red line precisely outline the area generated by the sample injection. (D) Yielding the area under the peak. By clicking on the Integrate button (C), the calculated area under the peak is displayed on the top right of the screen. <a href="https://www.jove.com/files/ftp_upload/63107/Supplemental Figure 2.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure 3: Representative spreadsheet with calibration curve and results. The calculated areas under the peaks generated from nitric oxide (NO) consumption by injection of samples with a known concentration of cell-free hemoglobin (expressed as [&#181;M] of heme) are shown on the top left (on yellow background). The values are plotted to build the calibration curve and its resulting linear equation y= mx + q. The slope (m) is the number of oxyHb heme groups (&#181;M) required to decrease the signal detected by the photomultiplier tube by 1 mV. Calculated values from triplicate injections (Run N 1,2,3) of samples from one patient obtained at four different time points (marked by different background colors) were entered in the spreadsheet. The average of each triplicate was used to yield the value of NO consumption caused by each sample by solving the linear equation: <a href="https://www.jove.com/files/ftp_upload/63107/Supplemental Figure 3.pdf" target="_blank">Please click here to download this File.</a>
<img alt="Equation 1" src="/files/ftp_upload/63107/63107eq01.jpg" />
Supplemental File 1: Reviewing the peaks using the bundled software. This file includes a screenshot taken from the bundled software and the actions allowed for each button. For every peak (including false peaks caused by artifacts), the user can use the buttons indicated in the file to confirm, or disregard, or delete, or name a specific peak, as needed. The user can also record a peak that was not previously detected. <a href="https://www.jove.com/files/ftp_upload/63107/Supplemental File 1.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds at JoVE.com

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

chemiluminescence-based-assays-for-detection-nitric-oxide-its

Universidad San Sebastian

Research

JoVE Journal

Medicine

3.7K Views.  Massachusetts General Hospital. Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity. 

Video: Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user&#8217;s specific biological and technical needs.

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

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Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice