Name: assessment of oxidative damage in the primary mouse ocular surface cells/stem cells in response to ultraviolet-c (uv-c) damage
Uploaded: 2020-02-15T12:00:00
Description: Watch this Scientific Journal Video about Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage at JoVE.com

The authors acknowledge support from the Yenepoya Research Centre, Yenepoya (Deemed to be University) for the infrastructural facilities.

The experiment was performed on primary ocular cells/stem cells derived from the Swiss albino mouse eye. The use of animals for harvesting the eyes for this experiment was approved by the Institutional Animal Ethical Committee, Yenepoya (Deemed to be University) (IEAC approval number, 6a/19.10.2016).
1. Preparations of reagents
NOTE: The derivation of primary cells/stem cells from the mouse ocular surface is beyond the scope of this protocol. Hence, we demonstrate the...

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	<li>Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ocular+surface:+the+challenge+to+enable+and+protect+vision:+the+Friedenwald+lecture.">The ocular surface: the challenge to enable and protect vision: the Friedenwald lecture.</a> Investigative Ophthalmology and Visual Science. 48 (10), 4391-4398 (2007).</li><li>Sridhar, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+cornea+and+ocular+surface.">Anatomy of cornea and ocular surface.</a> Indian Journal of Ophthalmoogy. 66 (2), 190-194 (2018).</li><li>Betteridge, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+oxidative+stress.">What is oxidative stress.</a> Metabolism. 49 (2), 3-8 (2000).</li><li>Ray, P. D., Huang, B. W., Tsuji, Y. <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+(ROS)+homeostasis+and+redox+regulation+in+cellular+signaling.">Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling.</a> Cell Signaling. 24 (5), 981-990 (2012).</li><li>Nita, M., Grzybowski, A. <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+the+Reactive+Oxygen+Species+and+Oxidative+Stress+in+the+Pathomechanism+of+the+Age-Related+Ocular+Diseases+and+Other+Pathologies+of+the+Anterior+and+Posterior+Eye+Segments+in+Adults.">The Role of the Reactive Oxygen Species and Oxidative Stress in the Pathomechanism of the Age-Related Ocular Diseases and Other Pathologies of the Anterior and Posterior Eye Segments in Adults.</a> Oxidative Medicine and Cellular Longevity. 2016, 3164734 (2016).</li><li>Covarrubias, L., Hernandez-Garcia, D., Schnabel, D., Salas-Vidal, E., Castro-Obregon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+reactive+oxygen+species+during+animal+development:+passive+or+active.">Function of reactive oxygen species during animal development: passive or active.</a> Developmental Biology. 320 (1), 1-11 (2008).</li><li>Behar-Cohen, F., 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=Ultraviolet+damage+to+the+eye+revisited:+eye-sun+protection+factor+(E-SPF(R)),+a+new+ultraviolet+protection+label+for+eyewear.">Ultraviolet damage to the eye revisited: eye-sun protection factor (E-SPF(R)), a new ultraviolet protection label for eyewear.</a> Clinical Ophthalmology. 8, 87-104 (2014).</li><li>Izadi, M., Jonaidi-Jafari, N., Pourazizi, M., Alemzadeh-Ansari, M. H., Hoseinpourfard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photokeratitis+induced+by+ultraviolet+radiation+in+travelers:+A+major+health+problem.">Photokeratitis induced by ultraviolet radiation in travelers: A major health problem.</a> Journal of Postgraduate Medicine. 64 (1), 40-46 (2018).</li><li>de Jager, T. L., Cockrell, A. E., Du Plessis, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+Light+Induced+Generation+of+Reactive+Oxygen+Species.">Ultraviolet Light Induced Generation of Reactive Oxygen Species.</a> Advances in Experimental Medicine and Biology. 996, 15-23 (2017).</li><li>Degl'Innocenti, D., 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=Oxadiazon+affects+the+expression+and+activity+of+aldehyde+dehydrogenase+and+acylphosphatase+in+human+striatal+precursor+cells:+A+possible+role+in+neurotoxicity.">Oxadiazon affects the expression and activity of aldehyde dehydrogenase and acylphosphatase in human striatal precursor cells: A possible role in neurotoxicity.</a> Toxicology. 411, 110-121 (2019).</li><li>Li, Z., 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=APC-Cdh1+Regulates+Neuronal+Apoptosis+Through+Modulating+Glycolysis+and+Pentose-Phosphate+Pathway+After+Oxygen-Glucose+Deprivation+and+Reperfusion.">APC-Cdh1 Regulates Neuronal Apoptosis Through Modulating Glycolysis and Pentose-Phosphate Pathway After Oxygen-Glucose Deprivation and Reperfusion.</a> Cellular and Molecular Neurobiology. 39, 123-135 (2019).</li></ol>

The DCFDA staining method described here enables the visualization of ROS in mouse primary ocular live cells treated with UV-C radiation. An advantage of this staining method is that it also allows the researchers to study the immediate effects of UV-C (3 hours post UVC exposure) on the live cells and their simultaneous enumeration for the percentage of ROS positive, as well as, dead cells. Moreover, as the staining method is used on the live cells, the cells can be further incubated in the same media for a longer time (...

The ocular surface (OS) is a functional unit mainly composed of the outer layer and glandular epithelia of cornea, lachrymal gland, meibomian gland, conjunctiva, part of the eye lid margins and innervations that transduce signals1. The transparent dome shaped corneal layer focuses light onto the retina. This avascular tissue is composed of cellular components such as epithelial cells, keratocytes, and endothelial cells and acellular components such as collagen and glycosaminoglycans2. The area is drained by tears that also supply most of the nutrients. The anatomical position of the OS compels it to be in direct contact with the external environment, often exposing it to various harsh components such as bright light, microbes, dust particles and chemicals. This factor predisposes the OS to physical injuries and makes it prone to various diseases.
Oxidative stress is caused due to the disequilibrium between the production of reactive oxygen species (ROS) and the endogenous antioxidant defenses mechanisms3. ROS are classified into reactive molecules and free radicals, both of which are derived from molecular oxygen (O2) through mitochondrial oxidative phosphorylation4. The former group is composed of non-radical species such as hydrogen peroxide (H2O2), singlet oxygen (1O2) and the latter includes species such as superoxide anions (O2-), and hydroxyl radicals (&#8226;OH), among others. These molecules are by-products of normal cellular processes and their roles have been implicated in important physiological functions such as signal transduction, gene expression, and host defense5. An enhanced production of ROS is known to be generated in response to factors such as pathogen invasion, xenobiotics, and exposure to ultra violet (UV) radiation4. This overproduction of ROS results in oxidative stress that leads to the damage of molecules such as nucleic acids, proteins, and lipids6.
Natural sunlight, the most predominant source of UV radiation, is composed of UV-A (400&#8211;320 nm), UV-B (320&#8211;290 nm), and UV-C (290&#8211;200 nm)7. An inverse correlation between the wavelength and spectral energies has been reported. Although natural UV-C radiations are absorbed by the atmosphere, artificial sources such as mercury lamps and welding instruments emit and, therefore, constitute an occupational hazard. Symptoms of exposure to eyes include photokeratitis and photokeratoconjunctivitis8. Production of ROS is one of the major mechanisms of inflicting UV induced cellular damage9. In the current study, we demonstrate the detection of ROS using the 2&#39;,7&#39;-Dichlorodihydrofluorescein diacetate (DCFDA) staining method in mouse primary ocular surface cells/stem cells exposed to UV-C. The green fluorescence was captured using fluorescent microscopy. Cells were counter-stained with two dyes, Hoechst 33342 and red propidium iodide, to stain the live and dead cells, respectively.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2&#39;,7&#39;-Dichlorofluorescein diacetate (DCFDA)</td>
			<td>Sigma</td>
			<td>D6883</td>
			<td>2&#39;,7&#39;-Dichlorofluorescein diacetate is fluorogenic probe and is permeable to cells. It is used for quantification of reactive oxygen species.</td>
		</tr>
		<tr>
			<td>Cell culture dish (35 mm)</td>
			<td>Eppendorf</td>
			<td>SA 003700112</td>
			<td>Sterile dishes for culturing the cells.</td>
		</tr>
		<tr>
			<td>DMEM High Glucose</td>
			<td>HiMedia</td>
			<td>AT007</td>
			<td>Most widely used cell culture media, contains 4500 mg/L of glucose.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, EU Origin</td>
			<td>HiMedia</td>
			<td>RM99955</td>
			<td>One of the most important components of cell culture media. It provides growth factors, amino acids, proteins, fat-soluble vitamins such as A, D, E, and K, carbohydrates, lipids, hormones, minerals, and trace elements.</td>
		</tr>
		<tr>
			<td>GlutMax</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Used as a supplement and an alternative to L-glutamine. It helps in improving cell viability and growth.</td>
		</tr>
		<tr>
			<td>HL-2000 Hybrilinker</td>
			<td>UVP</td>
			<td></td>
			<td>Hybridization oven/UV cross linker</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma</td>
			<td>B2261</td>
			<td>Hoechst stain is permeable to both live and dead cells. It binds to double starnded DNA irrespective of wether the cell is dead or alive.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td></td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (100X)</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td>Used as a supplement to increase the cell growth and viability.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (Pen-Strep)</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Penicillin and streptomycin is used to prevent the bacterial contamination in culture.</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td>Fluorescent dye which is only permeable to dead cells. It binds with DNA and helps in distinguishing between live and dead cells.</td>
		</tr>
		<tr>
			<td>TryplE Express</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Gentle cell dissociation agent</td>
		</tr>
		<tr>
			<td>ZOE Fluorescent Cell Imager</td>
			<td>Bio-rad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of oxidative damage in the primary mouse ocular surface cells/stem cells in response to ultraviolet-c (uv-c) damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

DCFDA is a colorless dye that is a chemically reduced form of fluorescein used as an indicator for detecting ROS in cells. This dye gets trapped inside cells and is easily oxidized to fluorescent dichlorodihydrofluorescein (DCF), which emits a green fluorescence. This fluorescence can be detected using fluorescent microscopy. The cells can be visualized and correlated with ROS accumulation as follows: (i) live cells without ROS emit high blue fluorescence; (ii) live cells with ROS accumulation emit high blue fluorescence...

Watch this Scientific Journal Video about Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage at JoVE.com

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

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