This work was supported by National Institutes of Health (CA142664).

1. Detection of ROS using carboxy-H2DCFDA
Carboxy-H2DCFDA is non-fluorescent but in the presence of ROS, when this reagent is oxidized, it becomes green fluorescent.
<ol>
 <li>Immediately prior to use, prepare a fresh stock solution of carboxy-H2DCFDA in sterile dimethylsulfoxide (DMSO) or 100% ethanol. Avoid multiple thaw/freeze cycles of your dye.</li>
 <li>Wash the cells with HEPES buffered salt solution (HBSS) or phosphate-buffered saline (PBS) to remove traces of the original medium.</li>
 <li>Load the cells with the dye (and the control dye, cf note section). In this assay we used Jurkat, a human leukemia cell line. Use carboxy-H2DCFDA at a final concentration of 1&mu;M in regular culture medium with reduced serum (2%).</li>
 <li>Incubate the cultures for 30 minutes in the dark, in a conventional incubator (37&deg;C, 5% CO2). Discard all unused dye solutions.</li>
 <li>Remove carboxy-H2DCFDA containing medium and wash twice with HBSS or PBS. From this step forward, protect your cells from light.</li>
 <li>Add fresh medium containing your drug of choice to the carboxy-H2DCFDA -loaded cells and incubate as desired. For this example we use H2O2 (0.03%) for 1 hour.</li>
 <li>Assess ROS by immediately analyzing your cells by flow cytometry using the FL1 channel (green fluorescence), or by fluorescence plate reader, or by fluorescence microscopy. The fluorescence can be detected by using excitation and emission wavelengths that are appropriate for green fluorescence.</li>
</ol>
Note: Controls should include carboxy-H2DCFDA-loaded untreated cells and unstained untreated cells. Carboxy-H2DCFDA is known to detect peroxides but could also be oxidized by other ROS. This reagent can also be modified by other means, oxidation insensitive control dye such as 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) should therefore be included in the test.
2. Measurement of nitric oxide (NO) production
You will need Sulfanilamide and N-1-napthylethylenediamine dihydrochloride (NED) solutions, and Nitrite standard. This assay is called the Griess assay.
NED solution : Make a 0.1% solution of N-1-napthylethylenediamine dihydrochloride diluted in sterile water.Sulfanilamide solution : Make a 1% solution of sulfanilamide diluted in 5% phosphoric acid. Nitrite Standard: Dilute the 0.1M standard stock sodium nitrite to 100&mu;M in sterile medium, do a serial dilution in the same medium.
Storage Conditions: Store chemicals as directed by manufacturer at room temperature. When reconstituted, NED and Sulfanilamide solutions are stored immediately after use at 4&deg;C, in the dark, and for a maximum of 3 months.
<ol>
 <li>Culture cells in a 96 well plate, use triplicates for each condition, and include proper controls according to your experiments.</li>
 <li>Treat cells to induce NO production. In our experiment we use lipopolysaccharide (LPS) (100 ng/ml) and recombinant IL-4 to treat our cells. In this protocol, we used RAW 264.7, a mouse macrophage cell line (Mouse leukaemic monocyte macrophage cell line).</li>
 <li>On the day of the assay, bring both reagents to room temperature.</li>
 <li>Spin your plate, collect cell supernatants and transfer 50&mu;l to a new 96 well plate. Prepare limiting dilution wells of your standard stock solution to make a standard curve.</li>
 <li>Add 50&mu;l of Sulfanilamide Solution to each sample and control well and mix well.</li>
 <li>Incubate at room temperature for 10 minutes in the dark.</li>
 <li>Add 50&mu;l of N-1-napthylethylenediamine dihydrochloride solution to each sample and control well and mix well.</li>
 <li>Incubate at room temperature for 10 minutes in the dark.</li>
 <li>Measure absorbance immediately using a plate reader with filter of wavelengths between 520nm and 550nm.</li>
</ol>
If you utilize different plate/dish sizes, use 1/1/1 volume for each solution and sample supernatant.
A violet color will appear in the positive wells. Results obtained with the standard will help you check the stability of your solutions.
3. Detection of ROS action: oxidized proteins
A different method to identify the production of ROS is to look at the end results by detecting the oxidation of proteins. Indeed, ROS modify glutathione, an antioxidant that is expressed in most cells and plays a protective role against ROS. Following oxidation by ROS, modification of the reduced glutathione (GSH) results in the sulfhydryl (thiol) group of its cysteine being linked to a second glutathione via a disulfide bridge. This leads to the formation of a dimerized protein (oxidized protein GSSG). GSH can be restored via modification of GSSG by the enzyme glutathione reductase. The increase in the GSSG/GSH ratio reflects oxidative stress. The following assay is based on the detection and quantification of these oxidized proteins. This method is not selective for specific ROS but rather detects the effects of NO, H2O2, O2- and other ROS. Here we measure the total amount of oxidized (GSSG) and reduced (GSH) glutathione using bioluminescent signals.
<ol>
 <li>Seed your cells in a 96 well plate (white/transparent, flat bottoms) as usual. For our experiment, we used 1x104 cells per well for adherent Raw 264.7 and A549 cells and suspension Jurkat cells. Depending on the size of your cells these numbers could be adjusted. We recommend to plate adherent cells the day before the test to allow them to attach to the plate. Enough wells should be prepared for oxidized and reduced protein detection as well as for "medium only" and untreated cells as controls. Use triplicates for all conditions.</li>
 <li>Treat your cells with your drug for the required time. Here we treated Jurkat cells and A549 cells for 1h with H2O2 (at 5mM and 2.5mM respectively). Raw 264.7 cells were treated with 200ng/ml of LPS for 16 h. During this incubation time, bring all your reagents to room temperature (RT). Make all reagents no more than 30 minutes before performing the test. The table below shows the volumes of reagents per well. When possible, it is important to remove the media containing the reducing agent before proceeding with the test.</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td rowspan="2"> </td>
 <td colspan="2">Adherent cells</td>
 <td colspan="2">Suspension cells</td>
 </tr>
 <tr>
 <td>Total Glu Lysis</td>
 <td>Oxidized Glu Lysis</td>
 <td>Total Glu Lysis</td>
 <td>Oxidized Glu Lysis</td>
 </tr>
 <tr>
 <td>NEM, 25mM</td>
 <td>none</td>
 <td>0.5&mu;l</td>
 <td>none</td>
 <td>0.5&mu;l</td>
 </tr>
 <tr>
 <td>Luciferin-NT</td>
 <td>1 &mu;l</td>
 <td>1 &mu;l</td>
 <td>1 &mu;l</td>
 <td>1 &mu;l</td>
 </tr>
 <tr>
 <td>Passive Lysis Buffer, 5X</td>
 <td>10&mu;l</td>
 <td>10&mu;l</td>
 <td>10&mu;l</td>
 <td>10&mu;l</td>
 </tr>
 <tr>
 <td>Distilled water</td>
 <td>39.0&mu;l</td>
 <td>38.5&mu;l</td>
 <td>14&mu;l</td>
 <td>13.5&mu;l</td>
 </tr>
 <tr>
 <td>Final volume per well</td>
 <td>50&mu;l</td>
 <td>50&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 </tr>
 </tbody>
</table>
<ol start="3">
 <li>Remove medium from wells with adherent cells. Do not remove medium in wells with suspension cells.</li>
 <li>Add reduced glutathione lysis reagent or oxidized glutathione lysis reagent to the corresponding wells and shake for 5 minutes at RT.</li>
 <li>Add the Luciferin generation reagent and incubate for 30 minutes at RT.</li>
 <li>Add the Luciferin detection reagent and incubate for 15 minutes at RT.</li>
 <li>Read the bioluminescent signal at integration time of 0.25-1 second per well using a plate reader luminometer.</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>
 </td>
 <td>
 Adherent cells</td>
 <td>
 Suspension cells</td>
 </tr>
 <tr>
 <td>Cell number per well</td>
 <td>1x104</td>
 <td>1x104</td>
 </tr>
 <tr>
 <td>Cell suspension per well+ drug</td>
 <td>100 &mu;l, to be removed before 3.4</td>
 <td>25&mu;l, not to be removed</td>
 </tr>
 <tr>
 <td>Reduced Glutathione Lysis Reagent</td>
 <td>50 &mu;l</td>
 <td>25&mu;l</td>
 </tr>
 <tr>
 <td>Oxidized Glutathione Lysis Reagent</td>
 <td>50 &mu;l</td>
 <td>25&mu;l</td>
 </tr>
 <tr>
 <td>Luciferin Generation Reagent</td>
 <td>50 &mu;l</td>
 <td>50 &mu;l</td>
 </tr>
 <tr>
 <td>Luciferin Detection Reagent</td>
 <td>100 &mu;l</td>
 <td>100 &mu;l</td>
 </tr>
 </tbody>
</table>
4. Representative Results:
<img alt="Figure 1" src="/files/ftp_upload/3357/3357fig1.jpg" /> 
Figure 1 Detection of ROS using carboxy-H2DCFDA dye. Jurkat cells (human leukemia cell line) treated with H2O2 were compared to non-treated cells. ROS induces the modification of carboxy-H2DCFDA that fluoresces green as detected by flow cytometry, the fluorescent peak in H2O2 treated cells shift compared to the peaks in controls (H2O2 treated cells stained with oxidation insensitive dye and non-treated cells stained with carboxy-H2DCFDA) . Results confirm the presence of ROS in treated cells.
<img alt="Figure 2" src="/files/ftp_upload/3357/3357fig2.jpg" /> 
Figure 2 Detection of NO using Griess reagents. RAW 264.7 cells (mouse macrophage) were treated with LPS and IL-4. A significant increased in NO production was detected in treated cells compared to control untreated cells.
<table border="1">
 <tbody>
 <tr>
 <td>
 Cell lines</td>
 <td>
 Non-treated</td>
 <td>
 Treated</td>
 </tr>
 <tr>
 <td>Raw 264.7</td>
 <td>13.0</td>
 <td>8.3</td>
 </tr>
 <tr>
 <td>A549</td>
 <td>21.6</td>
 <td>10.5</td>
 </tr>
 <tr>
 <td>Jurkat</td>
 <td>5.2</td>
 <td>2.8</td>
 </tr>
 </tbody>
</table>
Table 1 Detection of ROS mediated oxidation of proteins. RAW 264.7 cells were treated with LPS, Jurkat and A549 (human lung cancer) cells were treated with H2O2. Results are expressed as the ratio reduced (GSH)/oxidized (GSSG) glutathione. Lower ratios of glutathione (GSH)/(GSSG) were detected in treated cells compared to control untreated cells, revealing that proteins were more oxidized in treated cells.

<ol>
	<li>Guzik, T. J., Korbut, R., Adamek-Guzik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+superoxide+in+inflammation+and+immune+regulation.">Nitric oxide and superoxide in inflammation and immune regulation.</a> J Physiol Pharmacol. 54, 469-487 (2003).</li><li>Perl, A., Gergely, P., Banki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+T+cells+of+patients+with+systemic+lupus+erythematosus.">Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus.</a> Int Rev Immunol. 23, 293-313 (2004).</li><li>Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., Telser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+antioxidants+in+normal+physiological+functions+and+human+disease.">Free radicals and antioxidants in normal physiological functions and human disease.</a> Int J Biochem Cell Biol. 39, 44-84 (2007).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol Rev. 82, 47-95 (2002).</li><li>Nakamura, K., Yube, K., Miyatake, A., Cambier, J. C., Hirashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+CD4+D3-D4+membrane+proximal+extracellular+domain+for+the+inhibitory+effect+of+oxidative+stress+on+activation-induced+CD4+down-regulation+and+its+possible+role+for+T+cell+activation.">Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.</a> Mol Immunol. 39, 909-921 (2003).</li><li>Los, M., Droge, W., Stricker, K., Baeuerle, P. A., Schulze-Osthoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+as+a+potent+activator+of+T+lymphocyte+functions.">Hydrogen peroxide as a potent activator of T lymphocyte functions.</a> Eur J Immunol. 25, 159-165 (1995).</li><li>Deem, T. L., Cook-Mills, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+cell+adhesion+molecule+1+(VCAM-1)+activation+of+endothelial+cell+matrix+metalloproteinases:+role+of+reactive+oxygen+species.">Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species.</a> Blood. 104, 2385-2393 (2004).</li><li>Pacher, P., Beckman, J. S., Liaudet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+peroxynitrite+in+health+and+disease.">Nitric oxide and peroxynitrite in health and disease.</a> Physiol Rev. 87, 315-424 (2007).</li><li>Griendling, K. K., Sorescu, D., Lassegue, B., Ushio-Fukai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+kinase+activity+and+gene+expression+by+reactive+oxygen+species+and+their+role+in+vascular+physiology+and+pathophysiology.">Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.</a> Arterioscler Thromb Vasc Biol. 20, 2175-2183 (2000).</li><li>Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S., Tiganis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+enhance+insulin+sensitivity.">Reactive oxygen species enhance insulin sensitivity.</a> Cell Metab. 10, 260-272 (2009).</li><li>Goldstein, B. J., Mahadev, K., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+paradox:+insulin+action+is+facilitated+by+insulin-stimulated+reactive+oxygen+species+with+multiple+potential+signaling+targets.">Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets.</a> Diabetes. 54, 311-321 (2005).</li></ol>

We received the support of Promega for this publication.

Several pathological situations such as inflammatory diseases, cancers, ischemia/reperfusion, and also treatments such as radiation or chemotherapy (i.e cisplatin) induce ROS overproduction. Thus, detecting and measuring ROS levels is important in many basic, pre-clinical and clinical studies. However, ROS have very short half lives and could be complicated to detect. Here, we propose simple tests that are routinely used and widely accepted for the detection of free radical production in mammalian cells.
<p class="j...

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA)</td>
			<td>Molecular Probes</td>
			<td>C369</td>
			<td>control</td>
		</tr>
		<tr>
			<td>carboxy-H2DCFDA</td>
			<td>Molecular Probes</td>
			<td>C400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sulfanilamide</td>
			<td>Sigma</td>
			<td>S9251-100G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N-1-napthylethylenediamine dihydrochloride</td>
			<td>Sigma</td>
			<td>N9125-10G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrite standard</td>
			<td>Sigma</td>
			<td>237213-100G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GSH/GSSG-Glo Assay</td>
			<td>Promega</td>
			<td>V6612</td>
			<td>To quantify oxidized, reduced or oxidized/reduced glutathione</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

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.

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 ...

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69.8K Views.  Baylor College of Medicine. Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Video: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

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. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

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 ...

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

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

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

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

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...