Name: comet assay as an indirect measure of systemic oxidative stress
Uploaded: 2015-05-22T15:00:00
Duration: 8 min 23 s
Description: Watch this Scientific Journal Video about Comet Assay as an Indirect Measure of Systemic Oxidative Stress at JoVE.com

We would like to thank the LHW Stiftung, in Triesen Liechtenstein, for financially supporting our research on oxidative stress.

Studies on comet assay performed in the University of Basel, Department of Ophthalmology were approved by the Local Ethical Committee of Basel
1. Preparation of Reagents
<ol>
	<li>Prepare Dulbecco&#39;s Phosphate Buffered Saline (PBS) (Ca++, Mg++ free) solution by adding PBS (1 L packet) and 990 ml of dH2O to the media. Adjust the pH to 7.4. Store at RT.</li>
	<li>Prepare lysing solution: To prepare 1,000 ml add 2.5 M NaCl (146.1 g), 100 mM EDTA (37.2 g) and 10 mM Trizma base (1.2 g) in dH2O.</li>
	<li>Prepare electrophoresis buffer (300 mM NaOH/1 mM EDTA) by adding 30 ml of 10 M NaOH and 7.5 ml of 0.2M EDTA, q.s. to 1,500 ml dH2O and mix well. The total volume depends on the gel box capacity. Prior to use, measure the pH of the buffer to ensure a pH of &#62;13.</li>
	<li>Prepare neutralization buffer containing 0.4 M Tris (48.5 gm added to ~800 ml dH2O, adjust pH to 7.5), concentrated (&#62;10 M HCl) in 1,000 ml with dH2O. Store the solution at RT.</li>
	<li>Prepare staining solution. To Ethidium Bromide (EtBr; 10x Stock, 20 &#181;g/ml) add 10 mg of EtBr in 50 ml dH2O and store at RT. For 1x Stock - mix 1 ml with 9 ml dH2O. CAUTIOUS! Handle EtBr with adequate precaution as it is a known carcinogen.</li>
</ol>
2. Isolation of Leukocytes
<ol>
	<li>Obtain human blood samples (20 ml) with anti-coagulant such as heparin using venous puncture.</li>
	<li>Separate the lymphocytes using a density gradient cell separator such as Histopaque.
	<ol>
		<li>Briefly, dilute blood in 1:1 ratio with PBS or RPMI (without FBS) and layer over 600 &#181;l cell separator liquid.</li>
		<li>Centrifuge 1,800 x g for 20 min at 4 &#176;C. Aspirate the &#8216;buffy&#8217; coat into 3-5 ml of PBS/ RPMI and centrifuged at 300 x g for 10 min to pellet the lymphocytes.</li>
		<li>Retrieve lymphocytes from just above boundary between PBS (RPMI) and cell separator liquid, using a pipettor. Remove the leukocyte bands from the interface between plasma and the cell separator liquid layers of each tube and collect into one 50 ml tube.</li>
	</ol>
	</li>
	<li>Bring the total volume to 50 ml with cold Dulbecco&#8217;s Modified Eagle Medium (DMEM). Wash the cell suspension three times with DMEM at 300 x g for 10 min.</li>
	<li>Count the total numbers of cells using a Haemocytometer. Suspend the cells in PBS and aliquote into microcentrifuge tubes at 107 cells/tube.</li>
	<li>Pipet 0.4 ml of cells into a 5 ml plastic disposable tube. Add 1.2 ml 1% low-gelling-temperature agarose at 40 &#176;C. Mix and rapidly and pipet 1.2 ml of cell suspension onto the agarose-covered surface of a pre-coated slide. Avoid producing bubbles.</li>
	<li>Allow the agarose to gel for about 2 min. Be consistent with the time and temperature used for gelling, and ensure that agarose is fully set before submerging in lysis solution. After fully set, submerge into lysing solution at ~4 &#186;C.</li>
</ol>
3. Lysis and Electrophoresis
NOTE: The procedure described is for electrophoresis under pH &#62;13 alkaline conditions.
<ol>
	<li>After at least 2 hr at ~4 &#186;C, gently remove slides from the lysing Solution. Place slides side by side on the horizontal gel box near one end, sliding them as close together as possible.</li>
	<li>Fill the buffer reservoirs with freshly made pH &#62;13 electrophoresis buffer until the liquid level completely covers the slides (avoid bubbles over the agarose).</li>
	<li>Let slides sit in the alkaline buffer for 20 min to allow for unwinding of the DNA and the expression of alkali-labile damage.</li>
	<li>Turn on power supply to 25 volts (~0.75 V/cm) and adjust the current to 300 milliamperes by raising or lowering the buffer level. Electrophorese the slides for 30 min.</li>
	<li>Turn off the power. Gently lift the slides from the buffer and place on a drain tray. Drop wise coat the slides with neutralization Buffer. Allow the slides to sit for at least 5 min. Drain slides and repeat two more times.</li>
	<li>Stain the slides with 80 &#181;l 1x Ethidium Bromide (EtBr) and leave for 5 min and then dip in chilled distilled water to remove excess stain. Then place a coverslip over the slide and store them immediately. Store slides for a month at RT in dry conditions. 
	NOTE: Other DNA stains (e.g., SYBR-green) may also be used. Caution! wear protective gloves as most dyes are mutagenic.</li>
</ol>
4. Quantification of DNA Breaks
<ol>
	<li>To visualize the DNA damage, observe the EtBr-stained DNA slide under a 40X objective fluorescent microscope.</li>
	<li>Assess DNA damage quantitatively in the cells by measuring the length of DNA migration and the percentage of migrated DNA (Figure 1A). Quantify DNA damage (Figure 1B) by the Tail- moment and Olive-moment. Definitions of Tail-Moment and Olive &#8211;Moment have previously been given8-10. 
	NOTE: In our experience it&#39;s enough to stick by one of the parameters. The cells are photographed by setting the automated tool that scans over the microscope slide and photographs cells. Quantification of DNA breaks can be done by image analysis software packages. There are several different software packages that are custom made to capture the image of the cells and quantify DNA damage. We recommend using the same software package throughout the experiment. The lab technician identifies the intact cell or comet to be scored on the computer and clicks with the mouse on it. The comet or intact cell is then automatically scored.</li>
</ol>

<ol>
	<li>Ostling, O., Johanson, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrophoretic+study+of+radiation-induced+DNA+damages+in+individual+mammalian+cells.">Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.</a> Biochem Biophys Res Commun. 123 (1), 291-298 (1984).</li><li>Phillips, J. L., Singh, N. P., Lai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromagnetic+fields+and+DNA+damage.">Electromagnetic fields and DNA damage.</a> Pathophysiology. 16 (2-3), 79-88 (2009).</li><li>Dhawan, A., Bajpayee, M., Parmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comet+assay:+a+reliable+tool+for+the+assessment+of+DNA+damage+in+different+models.">Comet assay: a reliable tool for the assessment of DNA damage in different models.</a> Cell Biol Toxicol. 25 (1), 5-32 (2009).</li><li>Aiub, C. A., Pinto, L. F., Felzenszwalb, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-repair+genes+and+vitamin+E+in+the+prevention+of+N-nitrosodiethylamine+mutagenicity.">DNA-repair genes and vitamin E in the prevention of N-nitrosodiethylamine mutagenicity.</a> Cell Biol Toxicol. 25 (4), 393-402 (2009).</li><li>Bartsch, H., Nair, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+inflammation+and+oxidative+stress+in+the+genesis+and+perpetuation+of+cancer:+role+of+lipid+peroxidation,+DNA+damage,+and+repair.">Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair.</a> Langenbecks Arch Surg. 391 (5), 499-510 (2006).</li><li>Martin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+and+repair:+relevance+to+mechanisms+of+neurodegeneration.">DNA damage and repair: relevance to mechanisms of neurodegeneration.</a> J Neuropathol Exp Neurol. 67 (5), 377-387 (2008).</li><li>Choy, C. K., Benzie, I. F., Cho, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-mediated+DNA+strand+breaks+in+corneal+epithelial+cells+assessed+using+the+comet+assay+procedure.">UV-mediated DNA strand breaks in corneal epithelial cells assessed using the comet assay procedure.</a> Photochem Photobiol. 81 (3), 493-497 (2005).</li><li>Mozaffarieh, 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=Comet+assay+analysis+of+single-stranded+DNA+breaks+in+circulating+leukocytes+of+glaucoma+patients.">Comet assay analysis of single-stranded DNA breaks in circulating leukocytes of glaucoma patients.</a> Mol Vis. 14, 1584-1588 (2008).</li><li>Mozaffarieh, M., Konieczka, K., Hauenstein, D., Schoetzau, A., Flammer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half+a+pack+of+cigarettes+a+day+more+than+doubles+DNA+breaks+in+circulating+leukocytes.">Half a pack of cigarettes a day more than doubles DNA breaks in circulating leukocytes.</a> Tob Induc Dis. 8 (14), (2010).</li><li>Mozaffarieh, M., Schotzau, A., Josifova, T., Flammer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+ranibizumab+versus+photodynamic+therapy+on+DNA+damage+in+patients+with+exudative+macular+degeneration.">The effect of ranibizumab versus photodynamic therapy on DNA damage in patients with exudative macular degeneration.</a> Mol Vis. 15, 1194-1199 (2009).</li><li>Pertynska-Marczewska, M., Merhi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+Advanced+Glycation+End+Products+With+Cardiovascular+Disease+in+Menopausal+Women.">Relationship of Advanced Glycation End Products With Cardiovascular Disease in Menopausal Women.</a> Reprod Sci. , (2014).</li><li>Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+quantitation+of+low+levels+of+DNA+damage+in+individual+cells).">A simple technique for quantitation of low levels of DNA damage in individual cells).</a> Exp Cell Res. 175, 184-191 (1988).</li><li>Gaiv&#227;o, I., Sierra, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+comet+assay:+insights,+uses,+and+future+perspectives.">Drosophila comet assay: insights, uses, and future perspectives.</a> Front Genet. 5, 304 (2014).</li><li>Zhang, J., 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=DNA+damage+in+lens+epithelial+cells+and+peripheral+lymphocytes+from+age-related+cataract+patients.">DNA damage in lens epithelial cells and peripheral lymphocytes from age-related cataract patients.</a> Ophthalmic Res. 51 (3), 124-128 (2014).</li><li>Huet, S. A., Vasseur, L., Camus, S., Chesn&#233;, C., Fessard, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+comet+and+micronucleus+assays+in+metabolic+competent+HepaRG+cells+to+predict+in+vivo+genotoxicity.">Performance of comet and micronucleus assays in metabolic competent HepaRG cells to predict in vivo genotoxicity.</a> Toxicol Sci. 138 (2), 300-309 (2014).</li><li>Danson, S., Ranson, M., Denneny, O., Cummings, J., Ward, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+the+comet-X+assay+as+a+pharmacodynamic+assay+for+measuring+DNA+cross-linking+produced+by+the+novel+anticancer+agent+RH1+during+a+phase+I+clinical+trial.">Validation of the comet-X assay as a pharmacodynamic assay for measuring DNA cross-linking produced by the novel anticancer agent RH1 during a phase I clinical trial.</a> Cancer chemotherapy and pharmacology. 60, 851-861 (2007).</li><li>Moller, P., Moller, L., Godschalk, R. W., Jones, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+reduction+of+comet+assay+variation+in+relation+to+DNA+damage:+studies+from+the+European+Comet+Assay+Validation+Group.">Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group.</a> Mutagenesis. 25, 109-111 (2010).</li><li>Chan, S. W., Chevalier, S., Aprikian, A., Chen, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+of+mitochondrial+DNA+damage+and+copy+number+in+circulating+blood:+a+sensitive+approach+to+systemic+oxidative+stress.">Simultaneous quantification of mitochondrial DNA damage and copy number in circulating blood: a sensitive approach to systemic oxidative stress.</a> BioMed Res Int. , 157-547 (2013).</li><li>Sanchez-Quesada, J. L., Perez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+lipoproteins+as+biomarkers+of+cardiovascular+risk+in+diabetes+mellitus.">Modified lipoproteins as biomarkers of cardiovascular risk in diabetes mellitus.</a> Endocrinol Nutr. 60, 518-528 (2013).</li><li>Gandhi, G., Kaur, G., Nisar, U. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-sectional+case+control+study+on+genetic+damage+in+individuals+residing+in+the+vicinity+of+a+mobile+phone+base+station.">Cross-sectional case control study on genetic damage in individuals residing in the vicinity of a mobile phone base station.</a> Electromagn Biol Med. 9, 1-11 (2014).</li><li>Cabarkapa, 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=Protective+effect+of+dry+olive+leaf+extract+in+adrenaline+induced+DNA+damage+evaluated+using+in+vitro+comet+assay+with+human+peripheral+leukocytes.">Protective effect of dry olive leaf extract in adrenaline induced DNA damage evaluated using in vitro comet assay with human peripheral leukocytes.</a> Toxicol In Vitro. 28 (3), 451-456 (2014).</li></ol>

Swedish scientists Ostling and Johanson were the first in their field to use comet assay to quantify DNA damage in cells1. The neutral conditions that the two scientists used, however, only allowed the detection of double-stranded DNA breaks. Singh et al. later adapted the assay for use under alkaline conditions, which produced a sensitive version that could assess double and single-stranded DNA breaks and detect alkali-labile sites12. Since its initial development, the assay has been modified at various steps to make it suitable for assessing types of DNA damage in different cell types13-15.
As with all techniques, paying rigorous attention to technical details is important to obtain accurate results16. Based on our experience, best results are obtained if every methodological step&#8212;from solution preparation to comet quantification&#8212;is performed by expert laboratory technicians. The use of fresh and correctly prepared media, adequate pipetting techniques, exact timing and (as previously mentioned in the results section) the use of freshly prepared leukocytes are essential in order for lysis and gel electrophoresis to obtain accurate results17.
All the individual steps in this assay are equally important for obtaining reliable results.16 In general best results are obtained if every single methodological step from solution preparation till comet quantification is performed by expert lab technicians.Important seem the use of fresh and correctly prepared media, adequate pipetting techniques, exact timing and as already referred to in the results section the use of freshly prepared leukocytes for lysis and gel-electrophoresis to obtain adequate results from the assay17.
As do other methodologies, the comet assay technique has its advantages and drawbacks. Being a sensitive method, the assay is vulnerable to factors (e.g., UV light) which could augment DNA breaks and thus affect results. Any factor that may enhance oxidative stress, except that which is being researched, should be avoided. Stressors can increase level of oxidative stress not only in leukocytes but also in all other blood mediums and lymphocytes. As mentioned earlier there are many different and more direct ways of measuring oxidative stress18,19. The comet assay is one of many methods which has certain advantages. These include its relative low cost, the small number of cells required (&#60;10,000 cells) and thus the small samples of blood required per patient, the relative short period of time required to quantify DNA damage in cells (approximately 3 days), its sensitivity and its wide-spread applicability to asses DNA damage in different cell-types. In the past we choose to work with leukocytes since we had experience with this cell-type and it was applicable for the particular study planned. Another advantage of the comet assay is that it can be used to detect different types and levels of DNA damage and thus can be applied in various other areas of studies besides oxidative stress such as DNA repair studies, supplementation trials or genotoxicity studies.
Oxidative stress is now recognized as playing a critical role in the pathogenesis of multiple diseases. The comet assay method, while one of many ways of measuring oxidative stress18,19, is relatively simple, versatile and inexpensive. When mastered, this method can be used in all areas of medicine in which oxidative stress plays role 8-10,20,21.

Comet assay was first developed by Swedish scientists Ostling and Johanson1. DNA is a negatively charged molecule. During electrophoresis, smaller, damaged DNA fragments travel faster toward a positively charged anode2. The smaller the DNA fragments, the faster their migration toward the anode to form a typical &#8220;comet&#8221; with a &#8220;head&#8221; composed of intact, undamaged DNA and a &#8220;tail&#8221; composed of damaged DNA fragments3. Damaged DNA can be repaired by different mechanisms in the body4, which keep the generation of DNA breaks fairly balanced5. If, however, the balance is in favour of DNA breaks , the breaks can accumulate and will ultimately contribute to the development of a disease.
Our DNA suffers approximately 0.000165% damage at a given time6. Single stranded DNA breaks refer to a defect on one of the two strands of the DNA double helix whereas double-stranded DNA breaks are caused when both strands of the DNA double helix are damaged. There are various factors which may enhance the amount of DNA breaks at a given time such as the age of the cell, cigarette smoke, various drugs or oxidative stress7-9. If all factors leading to enhanced DNA breaks are kept constant, then the quantification of DNA breaks by means of comet assay is a good indirect parameter of oxidative stress.
The method of comet assay is used increasingly today in medicine including ophthalmology. One reason for this is that oxidative stress is more and more recognized as a pathogenetic mechanism for various diseases8-11 and comet assay is one method with which oxidative stress can indirectly be quantified.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Qualigens</td>
			<td>(CPW59)</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium EDTA</td>
			<td>HiMedia</td>
			<td>(RM1370)</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Sigma</td>
			<td>(E-8751)</td>
			<td></td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>(1077-1)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) (Ca++, Mg++ free)</td>
			<td>HiMedia</td>
			<td>(TS1006)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Ranbaxy Rankem</td>
			<td>(S0160)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>BDH-Merck</td>
			<td>(89021)</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>HiMedia</td>
			<td>(RM 845)</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Spectrochem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials required for gel electrophoresis</td>
		</tr>
		<tr>
			<td>Normal Melting Agarose (NMA)</td>
			<td>HiMedia</td>
			<td>(RM273)</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Melting Point Agarose (LMPA)</td>
			<td>Sigma</td>
			<td>(A9414)</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol - Qualigens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips (No. 1, 24 x 60 mm)</td>
			<td>Blue Star</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td>Tarsons</td>
			<td>(500010)</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettor and Tips</td>
			<td>Tarsons</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides, Conventional / Micro gel electrophoresis (MGE) slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

comet assay as an indirect measure of systemic oxidative stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Although the method of comet assay renders reproducible results, it can be influenced by a variety of factors. One such factor is whether or not the leukocytes are cryopreserved prior to lysis and gel-electrophoresis or whether this step is performed on freshly prepared leukocytes. Figure 2 shows that in Group 2, where leukocytes were cryopreserved prior to lysis and gel electrophoresis ss- DNA breaks were significantly higher, than in Group 1 where lysis and gel -electrophoresis was done on freshly prepared cells.
The assay can also be used to indirectly assess systemic oxidative stress. Table 1 shows the descriptive statistics for Tail- moment and Olive- moment in smokers and healthy subjects. The data reveals that smoking half a pack of cigarettes daily more than doubles ssDNA breaks in the circulating leukocytes9.
<img alt="Figure 1A" src="/files/ftp_upload/52763/52763fig1A.jpg" />
<img alt="Figure 1B" src="/files/ftp_upload/52763/52763fig1B.jpg" /> 
Figure 1.&#160;(A) Depicts the microscope used during comet assay analysis. (B) &#916; depicts a typical &#8220;comet&#8221; with a bright head and tail (damaged smaller DNA fragments). <a href="https://www.jove.com/files/ftp_upload/52763/52763fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52763/52763fig2.jpg" /> 
Figure 2. ssDNA breaks were significantly higher when leukocytes were cryopreserved (Group 2) than when freshly prepared leukocytes were used prior to lysis and gel-electrophoresis.
<img alt="Table 1" src="/files/ftp_upload/52763/52763table1.jpg" /> 
Table 1. Smokers had more than double the number of ssDNA breaks in comparison to age and sex matched healthy non-smokers.

Watch this Scientific Journal Video about Comet Assay as an Indirect Measure of Systemic Oxidative Stress at JoVE.com

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

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Biology

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...