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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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).

1. Prepare a stock solution of 10 mM DCFDA by dissolving 25 mg of DCFDA powder in 5.13 mL of DMSO. Aliquot 250 µL each in amber colored 1.5 mL tubes and store at -20 °C.
2. 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 µL in amber colored microcentrifuge tubes and store at 2-6 °C for up to 6 months. For longer term storage, store at -20 °C.
3. 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 °C.

2. Cell plating and UV-C radiation treatment

1. 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).
2. 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 °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% β-mercaptoethanol.
3. 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 µL) to remain in contact with the cells, just enough to cover them.
4. 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.
5. Expose the cells to different grades/doses of UV-C: 1 J/m², 100J/m², 1,000 J/m² and 10,000 J/m².
6. After the UV-C exposure, replace the lid of each of the dishes immediately and remove them from the UV-C source chamber.
7. 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.
8. Incubate the cells for 3 h in a 37 °C CO₂ incubator. Three hours of incubation post UV-C exposure is optimal to visualize and quantify the early effects.

3. Preparation of live-cell staining media

1. Prepare the staining media fresh during the last 15 min of the 3 h cell incubation post UV-C exposure.
2. Pre-warm 10 mL of the staining media containing 10% FBS-DMEM supplemented with 1% Pen-Strep to 37 °C.
3. Add 5 μL of 10 mM DCFDA; 5 μL of 10 mg/mL Hoechst solution and 200 μL of 1 mg/mL propidium iodide. The final concentrations of DCFDA, Hoechst and PI are 5 μM, 5 μg/mL and 20 μg/mL, respectively, in the 10 mL of staining media.

4. DCFDA staining of UV-C exposed mouse primary ocular cells

1. After 3 h of incubation of UV-C exposed mouse primary ocular cells at various doses, aspirate the media from the 35 mm dishes.
2. Replenish with 2 mL of freshly prepared DCFDA staining media to each of the dishes gently from the sides.
3. Incubate the cells with the staining media for 15 min in the dark in a 37 °C CO₂ incubator for live-cell staining.

5. Viewing of DCFDA (ROS), Hoechst and PI stained cells

1. After the completion of incubation, discard the staining media.
2. 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.

6. Quantification of stained cells (Hoechst-Blue, PI-Dead and Green-ROS) using imaging techniques

1. Export the images captured under the inverted fluorescent microscope/cell imager to ImageJ for quantification.
2. 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.
3. 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.
4. 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.
5. 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).

Results

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...

Discussion

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 (...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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