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',7'-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.
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',7'-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.
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 (•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 (400–320 nm), UV-B (320–290 nm), and UV-C (290–200 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',7'-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.
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).
2. Cell plating and UV-C radiation treatment
3. Preparation of live-cell staining media
4. DCFDA staining of UV-C exposed mouse primary ocular cells
5. Viewing of DCFDA (ROS), Hoechst and PI stained cells
6. Quantification of stained cells (Hoechst-Blue, PI-Dead and Green-ROS) using imaging techniques
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%–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).
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). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Component | Volume |
10% Fetal Bovine Serum containing DMEM | 9790 μL |
DCFDA stock solution | 5 µL |
Hoechst 33342 stock solution | 5 µL |
Propidium iodide stock solution | 200 µL |
Table 1: Reagents required for the preparation of staining solution.
Problem | Probable reasons | Troubleshooting | コメント |
No or very little ROS or PI positive cells are stained when the cells were treated with high to highest UV dose | i. Use of an old staining solution. | i. Use freshly prepared staining solution. | i. Earlier prepared staining solution leads to improper staining of the cells. |
ii. Overconfluent culture dish resulting in salvage of UVC damage by the cells. | 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. | ii. Overconfluent cells can salvage of UVC damage, and hence no or little ROS and PI positive cells will be visualized. |
Table 2: Troubleshooting.
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 µL 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 (•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 authors have nothing to disclose.
The authors acknowledge support from the Yenepoya Research Centre, Yenepoya (Deemed to be University) for the infrastructural facilities.
2',7'-Dichlorofluorescein diacetate (DCFDA) | Sigma | D6883 | 2',7'-Dichlorofluorescein diacetate is fluorogenic probe and is permeable to cells. It is used for quantification of reactive oxygen species. |
Cell culture dish (35 mm) | Eppendorf | SA 003700112 | Sterile dishes for culturing the cells. |
DMEM High Glucose | HiMedia | AT007 | Most widely used cell culture media, contains 4500 mg/L of glucose. |
Fetal Bovine Serum, EU Origin | HiMedia | RM99955 | 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. |
GlutMax | Gibco, Thermo Fisher Scientific | 35050061 | Used as a supplement and an alternative to L-glutamine. It helps in improving cell viability and growth. |
HL-2000 Hybrilinker | UVP | Hybridization oven/UV cross linker | |
Hoechst 33342 | Sigma | B2261 | 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. |
Matrigel | Corning | Basement membrane matrix | |
MEM Non-Essential Amino Acids (100X) | Gibco, Thermo Fisher Scientific | 11140050 | Used as a supplement to increase the cell growth and viability. |
Penicillin-Streptomycin (Pen-Strep) | Gibco, Thermo Fisher Scientific | 15140122 | Penicillin and streptomycin is used to prevent the bacterial contamination in culture. |
Propidium Iodide | Sigma | P4170 | Fluorescent dye which is only permeable to dead cells. It binds with DNA and helps in distinguishing between live and dead cells. |
TryplE Express | Thermo Fisher Scientific | Gentle cell dissociation agent | |
ZOE Fluorescent Cell Imager | Bio-rad |