This work was funded by NIH intramural grant to Dr Alan N. Schechter.

NOTE: Blood samples were obtained from NIH blood bank (IRB approved protocol: 99-CC-0168).
1. Blood Sample Preparation
NOTE: To avoid platelet activation, draw blood slowly and mix gently with citrate by inverting the tube several times.
<ol>
	<li>Platelet-rich plasma (PRP) preparation
	<ol>
		<li>Draw 30&#8211;50 mL of blood using a 20 G or larger diameter needle and add into a tube containing sodium citrate (3.8%) in a 1:9 ratio or acid citrate dextrose (ACD) (85 mM trisodium citrate, 66.6 mM citric acid, 111 mM glucose, pH 4.5) in a 1:6 ratio.</li>
		<li>Aliquot 5 mL of freshly collected whole blood into empty 15-mL conical tubes. Centrifuge whole blood at 120 x g for 10 min at room temperature.</li>
		<li>Carefully collect the supernatant containing the platelets (platelet-rich plasma, PRP) from the upper portion by a plastic transfer pipette. 
		NOTE: PRP from Step 1.1.3 is ready for use in experiments or for further processing to prepare washed platelets. The soft pellet obtained after centrifugation (Step 1.1.2) contains red blood cells and white cells and can be further purified to obtain washed red blood cells (RBC) &#8212; see Step 1.3. The experiment needs to be finished within 2 h after drawing blood. When collecting PRP (supernatant), avoid the collection of buffy coat-containing leukocytes accumulated on the PRP/RBC interface.</li>
	</ol>
	</li>
	<li>Washed platelet preparation (optional)
	<ol>
		<li>Centrifuge collected PRP at 400 x g for 10 min at room temperature. Discard the supernatant and keep the platelet pellets.</li>
		<li>Gently wash the pellets by adding 5 mL of CGS buffer (120 mM NaCl, 12.9 mM trisodium citrate and 30 mM glucose, pH 6.5) and centrifuge at 400 x g for 10 min at room temperature.</li>
		<li>Resuspend the platelet pellets with 3 mL of modified Tyrode buffer (134 mM NaCl, 0.34 mM NaH2PO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose pH 7.4).</li>
		<li>Dilute the platelets (1:100 &#8212; as example, 10 &#181;L of platelets in 990 &#181;L of Rees-Ecker solution) with Rees Ecker solution and count by hemocytometer.</li>
		<li>Adjust the density of platelets to 3 x 108 cells/mL by adding Tyrode buffer.</li>
		<li>Incubate the platelet suspension at 37 &#176;C for 1 h before starting the experiment. 
		NOTE: Washed platelets are stable for 5&#8211;8 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of washed red blood cells (RBC)
	<ol>
		<li>After the collection, centrifuge the soft pellet obtained in Step 1.1.3 at 2,500 x g for 10 min at room temperature.</li>
		<li>Discard the supernatant and wash the RBC pellet with 5 mL of PBS by centrifugation at 2,500 x g for 10 min at room temperature. Repeat this step for 3 times.</li>
		<li>Discard the PBS and use washed RBCs for further experiments.</li>
	</ol>
	</li>
</ol>
2. Deoxygenation
<ol>
	<li>Add 1 mL of the mixture of PRP (prepared in Step 1.1.4. or 1.2.4.) and RBCs (prepared in Step 1.3.) at the desired hematocrit into a polypropylene bottle closed with a rubber stopper. For example, at 20% hematocrit, add 200 &#181;L of RBCs to 800 &#181;L of PRP. 
	NOTE: Suggested hematocrits are from 0% up to 40%. Do not use a glass bottle or flask as platelets adhere to glass surface.</li>
	<li>Insert the needle connected to helium gas tank into the closed bottle and make the gas outlet by inserting a 26 G needle (Figure 1).</li>
	<li>Slowly swirl the bottle at room temperature. 
	NOTE: Do not allow He gas to bubble through blood preparation. Slow gas flow is preferable for this procedure, and excessively fast He gas flow leads to increased hemolysis. The most efficient gas flow needs to be determined by trial, as it highly depends on the geometry of the experimental setup.</li>
	<li>Periodically follow the deoxygenation process by measuring the partial oxygen pressure (pO2) using a CO-oximeter. 
	NOTE: In our hands, 10 min of deoxygenation decreases pO2 to 25 mmHg which is required for nitrite reduction by RBCs. Continuing deoxygenation did further reduce pO2 to 10 mmHg; however, it led to increased hemolysis and increases in cell-free hemoglobin (Figure 2).</li>
</ol>
3. Red Blood Cell Nitrite Reduction
<ol>
	<li>Add 1 mL of PBS (pH 7.4) into 0.0345 g of NaNO2 to make 500 mM stock solution of nitrite. Dilute 500 mM NaNO2 to 250 &#181;M NaNO2 (25x) by serial dilution in PBS (pH 7.4).</li>
	<li>Using a microsyringe, inject nitrite (40 &#181;L) through the septum into the deoxygenated sample of PRP and RBCs to achieve final concentrations of 10 &#181;M in total 1 mL of sample. 
	NOTE: In this experiment, the final concentrations of nitrite used is 10 &#181;M.</li>
	<li>Incubate the sample at 37 &#176;C for 10 min or longer. 
	NOTE: Adjust the exact duration of RBC incubation with nitrite needed for maximal nitrite reduction for different type of experiments.</li>
	<li>Remove the stopper and pipette 1 mL of the sample into a microcentrifuge tube.</li>
	<li>Centrifuge at 200 x g for 3 min at room temperature.</li>
	<li>Pipette 300 &#181;L of the platelet suspension from the upper portion of the supernatant and mix with ACD (pH 6.5) in a 1:9 ratio. Discard the RBC pellet.</li>
	<li>Centrifuge the platelet suspension at 500 x g for 4 min at room temperature.</li>
	<li>Add 80 &#181;L of ice-cold lysis buffer (150 mM NaCl, 50 mM Tris, 0.5% NP-40, pH 7.4) containing protease inhibitor cocktail III (1:500) to the platelet pellets.</li>
</ol>
4. Western Blotting of VASP
<ol>
	<li>Load 15 &#181;g of the protein from each sample to 2 separate 10% SDS-PAGE gels. 
	NOTE: Harsh stripping buffer, which contains b-mercaptoethanol, cannot remove P-VASPSer239 and VASP antibodies from the membrane; therefore, P-VASP Ser239 and VASP should be run separately.</li>
	<li>Run the gels at 120 V for 1.30 h and transfer to the nitrocellulose membrane.</li>
	<li>Block non-specific binding with 5% non-fat dry milk.</li>
	<li>Incubate the membrane with P-VASPSer239 (1:500), VASP (1:1,000) and GAPDH (1:1,000) for overnight at 4 &#176;C.</li>
	<li>Incubate horseradish peroxidase (HRP) secondary antibody with the membrane for 45 min at room temperature.</li>
	<li>Expose the membrane with an imager machine and quantify the band density using ImageJ.</li>
</ol>

<ol>
	<li>Huang, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+between+nitrite+and+deoxyhemoglobin.+Reassessment+of+reaction+kinetics+and+stoichiometry.">The reaction between nitrite and deoxyhemoglobin. Reassessment of reaction kinetics and stoichiometry.</a> Journal of Biological Chemistry. 280 (35), 31126-31131 (2005).</li><li>Cosby, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrite+reduction+to+nitric+oxide+by+deoxyhemoglobin+vasodilates+the+human+circulation.">Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.</a> Nature Medicine. 9 (12), 1498-1505 (2003).</li><li>Mo, E., Amin, H., Bianco, I. H., Garthwaite, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+a+cellular+nitric+oxide/cGMP/phosphodiesterase-5+pathway.">Kinetics of a cellular nitric oxide/cGMP/phosphodiesterase-5 pathway.</a> Journal of Biological Chemistry. 279 (5), 26149-26158 (2004).</li><li>Park, J. W., Piknova, B., Nghiem, K., Lozier, J. N., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+effect+of+nitrite+on+coagulation+processes+demonstrated+by+thromboelastography.">Inhibitory effect of nitrite on coagulation processes demonstrated by thromboelastography.</a> Nitric oxide. 40, 45-51 (2014).</li><li>Wajih, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+red+blood+cell+S-nitrosation+in+nitrite+bioactivation+and+its+modulation+by+leucine+and+glycose.">The role of red blood cell S-nitrosation in nitrite bioactivation and its modulation by leucine and glycose.</a> Redox Biology. 8, 415-421 (2016).</li><li>Akrawinthawong, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometric+analysis+of+the+inhibition+of+platelet+reactivity+due+to+nitrite+reduction+by+deoxygenated+erythrocytes.">A flow cytometric analysis of the inhibition of platelet reactivity due to nitrite reduction by deoxygenated erythrocytes.</a> PLoS One. 9 (3), e92435 (2014).</li><li>Friebe, A., Koesling, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+nitric+oxide-sensitive+guanylyl+cyclase.">Regulation of nitric oxide-sensitive guanylyl cyclase.</a> Circulation Research. 93 (2), 96-105 (2003).</li><li>Srihirun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+inhibition+by+nitrite+is+dependent+on+erythrocytes+and+deoxygenation.">Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation.</a> PLoS One. 7 (1), e30380 (2012).</li><li>Smolenski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+regulation+of+vasodilator-stimulated+phosphoprotein+serine+239+phosphorylation+in+vitro+and+in+intact+cells+using+a+phosphospecific+monoclonal+antibody.">Analysis and regulation of vasodilator-stimulated phosphoprotein serine 239 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody.</a> Journal of Biological Chemistry. 273 (32), 20029-20035 (1998).</li><li>Burkhart, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+comprehensive+and+quantitative+analysis+of+human+platelet+protein+composition+allows+the+comparative+analysis+of+structural+and+functional+pathways.">The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways.</a> Blood. 120 (15), e73-e82 (2012).</li><li>Parakaw, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+inhibition+and+increased+phosphorylated+vasodilator-stimulated+phosphoprotein+following+sodium+nitrite+inhalation.">Platelet inhibition and increased phosphorylated vasodilator-stimulated phosphoprotein following sodium nitrite inhalation.</a> Nitric oxide. 66, 10-16 (2017).</li><li>Srihirun, S., Piknova, B., Sibmooh, N., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylated+vasodilator-stimulated+phosphoprotein+(P-VASPSer239)+in+platelets+is+increased+by+nitrite+and+partially+deoxygenated+erythrocytes.">Phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets is increased by nitrite and partially deoxygenated erythrocytes.</a> PLoS One. 13 (3), e0193747 (2018).</li><li>Mal Cortese-Krott, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+soluble+guanylate+cyclase+in+RBCs:+preserved+activity+in+patients+with+coronary+artery+disease.">Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease.</a> Redox Biology. 14, 328-337 (2018).</li><li>Abel, K., Mieskes, G., Walter, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+the+focal+adhesion+protein+VASP+in+vitro+and+in+intact+human+platelets.">Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets.</a> FEBS letter. 370 (3), 184-188 (1995).</li></ol>

Dr. Alan Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. He receives royalties based on NIH licensing of these patents for clinical development but no other compensation.

Since platelets are easily activated, gentle handling of platelet-containing samples is required. Fast pipetting and vigorous shaking should be avoided. Platelet inhibitors such as prostacyclin (PGI2) can be used to prevent platelet activation; however, this may affect some signaling pathways inside the platelets. For the preparation of platelet pellets, we add ACD to the platelet suspensions and use low speed centrifugation.
Freshly prepared platelets in PRP have a limited life spa...

Platelets are small disc-shaped cell fragments derived from megakaryocytes that are crucial for blood clotting. The clotting cascade is initiated by various bioactive molecules (such as collagen or ADP), released after the injury of vascular wall. The blood clotting process can be modified, among various effectors by nitric oxide (NO). NO, naturally produced by mammalian cells, is one of the most versatile physiological signals. It acts as a potent vasodilator, neurotransmitter and immune modulator, to name a few of its many functions. In the bloodstream, NO also helps to regulate the extent of blood clotting by inhibiting platelet aggregation. One of the most likely sources of NO in the bloodstream is nitrite, an inorganic ion that has been shown to serve as a precursor of NO. Reacting with red blood cells (RBCs), nitrite is reduced to NO and deoxyHb is oxidized to methemoglobin (metHb)1. NO released from RBCs is vasoactive and causes vasorelaxation2. This nitrite reduction pathway is an alternate NO generation pathway, acting together with and complementing the classical NO generation path by endothelial nitric oxide synthase at hypoxic conditions.
Platelets themselves are not able to reduce nitrite into NO but are very sensitive to its presence. In intact platelets, NO in the nanomolar range increases cGMP (EC50 = 10 nM) and phosphorylation of VASP (EC50 = 0.5 nM)3. Therefore, platelets may serve as an excellent sensor of nitrite reduction by RBCs and NO release into blood. There are several methods that can directly measure the extent of platelet activation - such as aggregometry and thromboelastography (TEG)4,5. However, these methods require expensive specialized instrumentation and rather large amounts of material. It is also possible to monitor events downstream, after NO is released from RBCs, using the changes in platelet surface protein expression &#8211; such as P-selectin6. NO is also known to increase the amount of cGMP in the platelets7. Previously, we used cGMP to monitor NO release into blood after nitrite reduction by deoxygenated RBC8. This proved to be a very sensitive method; however, cGMP is a short-lived molecule and its detection involves extensive labor. Another possibility, described in the presented protocol, uses phosphorylation of the vasodilator-stimulated phospho (VASP)-protein to detect the presence of NO in blood. VASP is a substrate of protein kinase G activation, which is phosphorylated upon the interaction with NO through the sGC/cGMP pathway9. Detectable VASP phosphorylation occurs at very low NO concentrations, which could make platelets a very sensitive detector of NO presence in blood. VASP is highly expressed in platelets, but not in other blood cells, which allows to follow selectively the events involving platelets10.
The main goal of this protocol is to describe the method in detail for the detection of NO release in whole blood using its interaction with platelets by monitoring VASP phosphorylation11,12. The described method allows early detection of low NO concentrations - theoretically in the nanomolar range which makes the present protocol more sensitive than cGMP determination, due to the use of standard Western blot techniques achievable in most laboratory settings.

<table><tbody><tr><td>Tri-sodium citrate</td><td>Supply by NIH blood bank</td><td></td><td></td></tr><tr><td>Citric acid</td><td>Supply by NIH blood bank</td><td></td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7528-250G</td><td></td></tr><tr><td>NaCl; sodium chloride</td><td>Sigma</td><td>S-7653 1kg</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;; sodium phosphate monobasic, monohydrate</td><td>Mallinckrodt Chemical</td><td>7892-04</td><td></td></tr><tr><td>KCl; potassium chloride</td><td>Mallinckrodt Chemical</td><td>6858</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;; sodium bicarbonate</td><td>Mallinckrodt Chemical</td><td>7412-12</td><td></td></tr><tr><td>HEPES; N-[2-Hydroxyethyl]piperazine-N&#039;-[-ethanesulfonic acid]</td><td>Sigma</td><td>H3375-500g</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;(1 M); magnesium chloride</td><td>Quality Biology</td><td>351-033-721</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;; calcium chloride</td><td>Sigma</td><td>C5080-500G</td><td></td></tr><tr><td>Nalgene Narrow-mouth HDPE Economy bottles</td><td>Nalgene</td><td>2089-0001</td><td></td></tr><tr><td>Red septum stopper NO.29</td><td>Fisherbrand</td><td>FB57877</td><td></td></tr><tr><td>NaNO&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt;; sodium nitrite</td><td>Sigma</td><td>S2252-500G</td><td></td></tr><tr><td>TRIZMA Base; Tris[hydroxymethyl]aminomethane</td><td>Sigma</td><td>T8524-250G</td><td></td></tr><tr><td>NP-40; 4-Nonylphenyl-polyethylene glycol</td><td>Sigma</td><td>74385-1L</td><td></td></tr><tr><td>Protease inhibitor cocktail set III</td><td>Calbiochem</td><td>539134</td><td></td></tr><tr><td>Phospho-VASP (Ser239) antibody</td><td>Cell signaling technology</td><td>3114</td><td></td></tr><tr><td>VASP antibody</td><td>Cell signaling technology</td><td>3112</td><td></td></tr><tr><td>GAPDH (14C10) Rabbit mAb</td><td>Cell signaling technology</td><td>2118</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M-6250-10ml</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td><td>Jackson Immuno Research Laboratories</td><td>111-035-003</td><td></td></tr><tr><td>Clarity Western ECL Substrate</td><td>BIO-RAD</td><td>1705060-200ml</td><td></td></tr><tr><td>CO-oximeter (ABL 90 flex)</td><td>Radiometer</td><td></td><td></td></tr></tbody></table>

platelet-based detection of nitric oxide in blood by measuring vasp phosphorylation

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

Venous blood samples have pO2 values between 50-80 mmHg. Deoxygenation by helium rapidly decreases pO2 to 25 mmHg within 10 min. Increased deoxygenation time slightly further decreases pO2. However, increased time of deoxygenation also leads to significantly increased levels of cell-free hemoglobin (determined by CO-Oximeter, visually seen on Figure 2 as increasingly red coloration of plasma) (Figu...

Watch this Scientific Journal Video about Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation at JoVE.com

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

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

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

platelet-based-detection-nitric-oxide-blood-measuring-vasp

Research

JoVE Journal

Medicine

7.9K Views.  Mahidol University. Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

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

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 ...

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.

Capturing dynamic changes in the protein activation of enucleated red blood cells poses methodological challenges, like the preservation of dynamic changes to acute stimuli for later assessment. The presented protocol describes sample preparation and staining techniques that enable preservation and analysis of relevant protein changes and subsequent detection.

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute ...

Biochemistry

Detection of Superoxide Anion in Platelets Using Fluorescence Imaging and EPR

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

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

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

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

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

Immunology and Infection

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Biology

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

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.

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 ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

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

Summary

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Chapters in this video

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

Two-photon Imaging of Intracellular Ca²⁺ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

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

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca²⁺]_iand Nitric Oxide Production

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch