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 UV-C exposure doses, reagent preparation for assessing ROS, live and dead cells and their quantification. Please refer to Table 1 for the respective volumes of the reagents (10% fetal bovine serum, DCFDA, Hoechst and propidium iodide stock solutions to be added for obtaining the final staining solution).
<ol>
	<li>Prepare a stock solution of 10 mM DCFDA by dissolving 25 mg of DCFDA powder in 5.13 mL of DMSO. Aliquot 250 &#181;L each in amber colored 1.5 mL tubes and store at -20 &#176;C.</li>
	<li>Prepare a stock solution of 10 mg/mL (16.23 mM solution) Hoechst 33342 by dissolving the entire contents of the 25 mg vial in 2.5 mL of deionized water. Make aliquots of 100 &#181;L in amber colored microcentrifuge tubes and store at 2-6 &#176;C for up to 6 months. For longer term storage, store at -20 &#176;C.</li>
	<li>Prepare a stock solution of 1 mg/mL propidium iodide in deionized water, aliquot 1 mL each in amber colored 1.5 mL tubes, and store at 4 &#176;C.</li>
</ol>
2. Cell plating and UV-C radiation treatment
<ol>
	<li>Before plating, dissociate the mouse primary ocular surface cells isolated in our laboratory (unpublished results; such cells are a mixture of corneal epithelial, stromal cells and keratocytes) using a gentle cell dissociation agent (Table of Materials).</li>
	<li>Plate 0.2 x 106 mouse primary ocular surface cells in 35 mm 0.2% basement membrane matrix coated cell culture dishes in 2.5 mL of complete media. Incubate overnight at 37 &#176;C in a 5% CO2 and humidified incubator. 
	NOTE: Complete media for culturing primary cells from the mouse ocular surface is comprised of DMEM high glucose containing 20% FBS, 1% Pen-strep, 1% Glutamax, 1% non-essential amino acid (NEAA), 1% sodium pyruvate, and 0.1% &#946;-mercaptoethanol.</li>
	<li>Before exposing the cells to various doses of UV-C, discard the maximum volume of media and allow only a thin layer of media (~500 &#181;L) to remain in contact with the cells, just enough to cover them.</li>
	<li>Take the dishes, one at a time to the UV-C source/chamber (the lower chamber of a hybridization oven/UV cross linker; Table of Materials). Place the dish into the chamber and remove the lid of the dish. The open lid position of the dish ensures that the cells receive the maximum UV-C dose during the UV-C exposure.</li>
	<li>Expose the cells to different grades/doses of UV-C: 1 J/m2, 100J/m2, 1,000 J/m2 and 10,000 J/m2.</li>
	<li>After the UV-C exposure, replace the lid of each of the dishes immediately and remove them from the UV-C source chamber.</li>
	<li>Bring each of the dishes to the laminar air flow hood and top up each of the dishes with 2 mL of fresh complete media.</li>
	<li>Incubate the cells for 3 h in a 37 &#176;C CO2 incubator. Three hours of incubation post UV-C exposure is optimal to visualize and quantify the early effects.</li>
</ol>
3. Preparation of live-cell staining media
<ol>
	<li>Prepare the staining media fresh during the last 15 min of the 3 h cell incubation post UV-C exposure.</li>
	<li>Pre-warm 10 mL of the staining media containing 10% FBS-DMEM supplemented with 1% Pen-Strep to 37 &#176;C.</li>
	<li>Add 5 &#956;L of 10 mM DCFDA; 5 &#956;L of 10 mg/mL Hoechst solution and 200 &#956;L of 1 mg/mL propidium iodide. The final concentrations of DCFDA, Hoechst and PI are 5 &#956;M, 5 &#956;g/mL and 20 &#956;g/mL, respectively, in the 10 mL of staining media.</li>
</ol>
4. DCFDA staining of UV-C exposed mouse primary ocular cells
<ol>
	<li>After 3 h of incubation of UV-C exposed mouse primary ocular cells at various doses, aspirate the media from the 35 mm dishes.</li>
	<li>Replenish with 2 mL of freshly prepared DCFDA staining media to each of the dishes gently from the sides.</li>
	<li>Incubate the cells with the staining media for 15 min in the dark in a 37 &#176;C CO2 incubator for live-cell staining.</li>
</ol>
5. Viewing of DCFDA (ROS), Hoechst and PI stained cells
<ol>
	<li>After the completion of incubation, discard the staining media.</li>
	<li>Add fresh complete media to the cells and observe the cells under an inverted fluorescent microscope/cell imager (Table of Materials). Photograph the desired fields: bright-field, blue fluorescence, red fluorescence, green fluorescence. 
	NOTE: The blue fluorescently stained cells are the nuclei, the green fluorescence is for ROS generating cells and the red fluorescence indicates the PI positive dead cells.</li>
</ol>
6. Quantification of stained cells (Hoechst-Blue, PI-Dead and Green-ROS) using imaging techniques
<ol>
	<li>Export the images captured under the inverted fluorescent microscope/cell imager to ImageJ for quantification.</li>
	<li>Open each of the images one at a time, using each channel (i.e., blue (Nuclei/Hoechst), green (ROS), red (Dead/PI positive)) sequentially, for counting. Start from the unexposed control and sequentially move to 1, 100, 1,000 and 10,000 J/m-2.</li>
	<li>Count the cells using the cell counting tool marked as a cross in the software menu for each of the fields [blue positive (Hoechst positive; nuclei), red positive (PI positive; dead cells); green positive (ROS)] in each of the images corresponding to each of the treatments.</li>
	<li>Count by clicking on each of the specific signals in each of the fields. For example, clicking on the blue/Hoechst stained nuclei will give the total number of nuclei in a given field.</li>
	<li>Calculate the results as the percentage of cell death by UV damage (number of PI positive cells x 100 divided by the number of Hoechst positive cells) and the percentage of ROS production by UV damage (number of DCFDA positive cells x 100 divided by the number of Hoechst positive cells).</li>
</ol>

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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>

Authors received funding support from Bio-Rad Laboratories India Private Limited for sponsoring the article.

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 (several days) for the study of delayed effects of UV-C radiation. Hence, this staining method makes it possible to visualize the ROS generation (green) and simultaneously distinguish the live, apoptotic and dead cell populations in live cells under an inverted fluorescence microscope. However, under certain circumstances, despite the anticipation of obtaining ROS positive and PI positive cells, the visualization can fail. For such instances, troubleshooting is suggested (Table 2).
This protocol must be carried out accurately to obtain optimal results. Optimal results indicate a correlation of the green fluorescent signal emitted/ROS generated as an accurate readout of the specific UV-C dose induced DNA damage. In other words, for carrying out this step, the volume of media that is in contact with the cells during the UV-C exposure should not be so much so that the UV-C dose fails to elicit the required damage. Hence, it is essential to keep a minimal volume of media, say 500 &#181;L per 35 mm dish, just enough to cover the cells during the UV-C exposure at all the specified doses. Secondly, the volume of media should not be so little that the cells dry up during UV-C exposure. Finally, immediately after the exposure of the cells to the UV-C, the cells should be topped up with fresh media to the optimal volume (say 2 mL), in order to further avoid any damages and ROS generation.
One limitation of this technique is the type of ROS generated. Other additional assays must be performed to detect the specific ROS type in response to the UV-C dose ranges. Such assays include the assessment of the intracellular hydroxyl radical (&#8226;OH), intracellular superoxide radicals, intracellular reactive nitrogen species, mitochondrial hydroxyl radicals, mitochondrial superoxides, and hydrogen peroxide in live cells using commercially available kits. Another limitation of this technique is the detectability of the percentage ROS generation at doses below 10 J/m2 and above 10,000 J/m2. Apparently, no ROS positive cells were visible when the primary cells/stem cells from the mouse ocular surface were exposed to UV-C doses less than 10 J/m2. On the contrary, 100% ROS positive cells were visible when the cells were exposed to UV-C dose above 10,000 J/m2. Hence, other assays (e.g., enzyme analysis, measurement of oxidative stress markers such as 8-hydroxydeoxyguanosine (8-OHdG) (DNA damage marker), and western blot analysis of DNA damage proteins at various doses) might be useful to understand the extent/type of cellular/molecular damages at various levels. A third limitation of this technique is the correlation of the UV-C doses to the ROS generated across various cell types. This needs to be validated.
Currently, DCFDA kits are available commercially for ROS detection either using microscopy or flow cytometry10,11. However, such kits are expensive and cannot be afforded by research laboratories in resource restricted countries. Hence, this protocol is very useful with an efficiency akin to the commercially sold methods. Secondly, we have incorporated the propidium iodide dead cell stain along with the DCFDA/ROS generation. Hence, live cell monitoring of ROS positive and dead cells can be carried out simultaneously using this method.

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><tbody><tr><td>2&#039;,7&#039;-Dichlorofluorescein diacetate (DCFDA)</td><td>Sigma</td><td>D6883</td><td>2&#039;,7&#039;-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 with low green fluorescence; and (iii) dead cells with ROS accumulation emit low blue fluorescence with high red and high green fluorescence.
Control cells and cells exposed to 1 J/m2 of UV-C did not exhibit DCFDA/ROS and PI positive cells. The UV-C unexposed control showed nuclear staining only as indicated by the Hoechst staining (Figure 1). However, no PI or DCFDA positive cells were seen in the UV-C unexposed control cells (Figure 1).
Cells exposed to 100 J/m2 of UV-C exhibited low ROS and PI positive cells. Primary cells from the mouse ocular surface upon exposure to UV-C at a dose of 100 J/m2 showed about 5% co-staining of DCFDA and PI, thereby, indicating the formation of ROS and cell death in 5% of the cells (Figure 1).
Cells exposed to 1,000 J/m2 of UV-C exhibited 60%&#8211;70% ROS and PI positive cells. Primary cells from mouse ocular surface upon exposure to UV-C at a dose of 1,000 J/m2 showed about 70% co-staining of DCFDA and PI, thereby, indicating the formation of ROS and cell death in 70% of the cells (Figure 1).
Cells exposed to 10,000 J/m2 of UV-C exhibited 100% ROS positive cells and ~100% cell death. The highest UV-C dose (10,000 J/m2) when exposed to the mouse ocular primary cells resulted in 100% cell death (PI positive cells) and ROS formation (DCFDA staining), thereby indicating the highest lethality of this particular UV-C dose (Figure 1).
Cells treated with 1 J/m2 of UV-C radiation did not show any accumulation of ROS and the percentage of live cells in this dose was comparable with that of the control cells. Cells demonstrated a significant amount of cell death and ROS accumulation at 100 J/m2. The highest amount of ROS accumulation and cell death was observed in cells treated with 10,000 J/m2 of UV-C radiation.
The quantified results (percentage of ROS generation and percentage of cell death as per the formulae given under section 6.3) were plotted in the form of a bar graph (Figure 2). The X-axis represents the UV-C doses while the Y-axis represents the percentage of cells. The green bar represents the percentage of ROS, and the red bars represent the cell death at different doses of UV-C radiation (Figure 2). The percentage of ROS and dead cells were of the order of 0%, 0%, 10%, 70% and 100% at the UV-C doses: unexposed control, 1 J/m2, 100 J/m2, 1,000 J/m2 and 10,000 J/m2, respectively (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59924/59924fig01.jpg" /> 
Figure 1: Composite live-cell images of the mouse ocular surface primary cells exposed to various doses of UV-C (Unexposed control, 1, 100, 1,000, 10,000 J/m2). These images were captured under different filters: bright-field (for cell morphology), blue (Hoechst nuclear stain), green (ROS generation) and red (propidium iodide stained dead cells). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59924/59924fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59924/59924fig02.jpg" /> 
Figure 2: Bar graphs showing the quantification results obtained after calculating the percentage of ROS generation and percentage of dead cells upon exposure of primary mouse ocular surface cells to various doses of UV-C radiation. The X-axis represents the UV-C doses, while the Y-axis represents the percentage of cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59924/59924fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>10% Fetal Bovine Serum containing DMEM</td>
			<td>9790 &#956;L</td>
		</tr>
		<tr>
			<td>DCFDA stock solution</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>Hoechst 33342 stock solution</td>
			<td>5 &#181;L</td>
		</tr>
		<tr>
			<td>Propidium iodide stock solution</td>
			<td>200 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Reagents required for the preparation of staining solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Probable reasons</td>
			<td>Troubleshooting</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td rowspan="2">No or very little ROS or PI positive cells are stained when the cells were treated with high to highest UV dose</td>
			<td>i. Use of an old staining solution.</td>
			<td>i. Use freshly prepared staining solution.</td>
			<td>i. Earlier prepared staining solution leads to improper staining of the cells.</td>
		</tr>
		<tr>
			<td>ii. Overconfluent culture dish resulting in salvage of UVC damage by the cells.</td>
			<td>ii. Plate only 0.2 million cells in each 35 mm cell culture dish and never allow the cells to grow for more than 12 h before exposing them to UVC doses.</td>
			<td>ii. Overconfluent cells can salvage of UVC damage, and hence no or little ROS and PI positive cells will be visualized.</td>
		</tr>
	</tbody>
</table>
Table 2: Troubleshooting.

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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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6.1K Views.  Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangaluru-575018, Karnataka, India. Hallo allemaal, mijn naam is Dr. Bipasha Bose. Ik werk als universitair hoofddocent en verantwoordelijk voor het Stem Cells and Reerative Medicine Center, Yenepoya Research Center, Yenepoya University, Mangalore, India. Nu gaan we laten zien hoe te detecteren voor de aanwezigheid voor ROS dat wil zeggen, reactieve zuurstof soorten "in de levende culturen van de muis oculaire oppervlakken die zijn blootgesteld aan verschillende doses van ultraviolet-C straling.Het voordeel van deze tech...

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

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H2DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H2DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H2DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H2DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.

A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

Neuroscience

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental 2-4 and epidemiological evidence 5,6 that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized 7 and non-anesthetized animals 8.
The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% 9. To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals 10. Our focus has recently been apoptosis in the lens as the mechanism for cataract development 8,11,12. It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract.
In this article, we describe how cataract can be experimentally induced by in vivo exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

Cataract is the leading cause of blindness in the world. Solar ultraviolet radiation (UVR) is the main risk factor for cataract development. An animal model of far UVR-B induced cataract was developed. In this article we describe methods for investigation of cataract formation: exposure to UVR, quantitative RT-PCR and immunohistochemistry.

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

Medicine

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Genetics

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

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.

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 ...

Biology

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical exposures, among other environmental factors, are an established cause of cataracts. Ocular toxicity testing can assess whether pharmaceuticals and their components may contribute to lens damage that may lead to cataracts or aid the treatment of cataracts. 
In vitro studies and in vivo animal testing can be used for assessing the safety of chemicals prior to clinical studies. The Draize test-the current in vivo standard for ocular toxicity and irritancy testing-has been criticized for lack of sensitivity and objective measurements of determining ocular toxicity. In vitro cell-based assays are limited as cell cultures cannot appropriately model an intact functional lens. 
The method described here is a sensitive in vitro alternative to animal testing, designed to evaluate the response of the intact bovine lens to treatment at both the cellular activity level and for overall refractive performance. The non-toxic reagent resazurin is metabolized in proportion to the level of cell activity. The lens laser-scanner assay measures the ability of the lens to refract incident beams of light to a single point with minimal error, directly relevant to its natural function. The method may be used to determine both acute and delayed changes in the lens, as well as the recovery of the lens from chemical or environmental exposures.

The goal of this protocol is to evaluate changes in metabolic activity and refractive function of the lens in response to experimental treatment.

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical ...

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

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