Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 ....

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.......

Representative 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 (.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
2',7'-Dichlorofluorescein diacetate (DCFDA)SigmaD68832',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)EppendorfSA 003700112Sterile dishes for culturing the cells.
DMEM High GlucoseHiMediaAT007Most widely used cell culture media, contains 4500 mg/L of glucose.
Fetal Bovine Serum, EU OriginHiMediaRM99955One 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.
GlutMaxGibco, Thermo Fisher Scientific35050061Used as a supplement and an alternative to L-glutamine. It helps in improving cell viability and growth.
HL-2000 HybrilinkerUVPHybridization oven/UV cross linker
Hoechst 33342SigmaB2261Hoechst stain is permeable to both live and dead cells. It binds to double starnded DNA irrespective of wether the cell is dead or alive.
MatrigelCorningBasement membrane matrix
MEM Non-Essential Amino Acids (100X)Gibco, Thermo Fisher Scientific11140050Used as a supplement to increase the cell growth and viability.
Penicillin-Streptomycin (Pen-Strep)Gibco, Thermo Fisher Scientific15140122Penicillin and streptomycin is used to prevent the bacterial contamination in culture.
Propidium IodideSigmaP4170Fluorescent dye which is only permeable to dead cells. It binds with DNA and helps in distinguishing between live and dead cells.
TryplE ExpressThermo Fisher ScientificGentle cell dissociation agent
ZOE Fluorescent Cell ImagerBio-rad

References

  1. Gipson, I. K. The ocular surface: the challenge to enable and protect vision: the Friedenwald lecture. Investigative Ophthalmology and Visual Science. 48 (10), 4391-4398 (2007).
  2. Sridhar, M. S. Anatomy of cornea and ocular surfac....

Explore More Articles

Oxidative DamagePrimary Mouse Ocular Surface CellsStem CellsUltraviolet C UV C DamageReactive Oxygen Species ROSDCFDAPropidium IodideHoechstLive Cell PermeantDead Cell PermeantUV CrosslinkerDose response

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved