Original Contributions
Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader1

https://doi.org/10.1016/S0891-5849(99)00107-0Get rights and content

Abstract

Oxidative stress (OS) has been implicated in various degenerative diseases in aging. In an attempt to quantify OS in a cell model, we examined OS induced by incubating for 30 min with various free radical generators in PC12 cells by using the dichlorofluorescein (DCF) assay, modified for use by a fluorescent microplate reader. The nonfluorescent fluorescin derivatives (dichlorofluorescin, DCFH), after being oxidized by various oxidants, will become DCF and emit fluorescence. By quantifying the fluorescence, we were able to quantify the OS. Our results indicated that the fluorescence varied linearly with increasing concentrations (between 0.1 and 1 mM) of H2O2 and 2,2′-azobios(2-amidinopropane) dihydrochloride (AAPH; a peroxyl radical generator). By contrast, the fluorescence varied as a nonlinear response to increasing concentrations of 3-morpholinosydnonimine hydrochloride (SIN-1; a peroxynitrite generator), sodium nitroprusside (SNP; a nitric oxide generator), and dopamine. Dopamine had a biphasic effect; it decreased the DCF fluorescence, thus acting as an antioxidant, at concentrations <500 μM in cells, but acted as a pro-oxidant by increasing the fluorescence at 1 mM. While SNP was not a strong pro-oxidant, SIN-1 was the most potent pro-oxidant among those tested, inducing a 70 times increase of fluorescence at a concentration of 100 μM compared with control. Collectively, due to its indiscriminate nature to various free radicals, DCF can be very useful in quantifying overall OS in cells, especially when used in conjunction with a fluorescent microplate reader. This method is reliable and efficient for evaluating the potency of pro-oxidants and can be used to evaluate the efficacy of antioxidants against OS in cells.

Introduction

Oxidative stress (OS) has been implicated in various degenerative diseases in aging such as atherosclerosis, cancer, Parkinson’s disease, and Alzheimer’s disease [1]. In order to understand the mechanisms of these diseases that involve OS and to implement treatment or prevention, an analytic method to evaluate OS in living cell models is very important. Although there are various methods to assess oxidative damage of cells, such as measuring lipid peroxidation products and DNA adducts [2], none of them evaluate the OS directly.

With the first description of using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as a fluorometric assay for hydrogen peroxide [3], it became popular to use dichlorofluorescin (DCFH) as a probe to evaluate intracellular hydrogen peroxide formation by flow cytometry. The theory behind using DCFH-DA is that nonfluorescent fluorescin derivatives will emit fluorescence after being oxidized by hydrogen peroxide [4]. The emitted fluorescence is directly proportional to the concentration of hydrogen peroxide. When applied to intact cells, the nonionic, nonpolar DCFH-DA crosses cell membranes and is hydrolyzed enzymatically by intracellular esterases to nonfluorescent DCFH [4], [5]. In the presence of reactive oxygen species (ROS), DCFH is oxidized to highly fluorescent dichlorofluorescein (DCF) [4]. Therefore, the intracellular DCF fluorescence can be used as an index to quantify the overall OS in cells.

Many studies have used fluorescent microscopy to quantify OS in cells using DCFH-DA. This method has the problem of inducing photooxidation of DCFH to DCF intracellularly and emitting fluorescence [6], because it is difficult to control the time of light exposure when trying to locate and focus cells under the microscope. In addition to the problem of photooxidation, there is no standard way to quantify the OS using a microscope. In order to prevent the overestimation of the OS due to photooxidation, an instrument equipped with fast light excitation and fast fluorescence capturing, which will not induce excessive photooxidation, is needed.

In this study, we used a fluorescent microplate reader to evaluate OS in cells, induced by applying various free radical generators extracellularly, using DCFH as the probe. Our results indicated that various free radical generators produced concentration-dependent changes in DCF fluorescence, indicating the indiscriminate nature of DCF. This feature of DCF allows the assay to be used to measure the overall OS in cells. By using a microplate reader, we were able to use 96-well plates, which gave us a large amount of data with low variability.

Section snippets

Cell cultures

PC12 cells were a gift from Dr. Arthur Tischler (Tufts University, School of Medicine, Boston, MA, USA). They were grown in growth medium containing 85% RPMI-1640 with l-glutamine, 10% heat-inactivated horse serum, 5% fetal bovine serum (FBS), 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate. The cells were maintained in collagen-coated plates in 5% CO2/95% air at 37°C. The culture medium was changed twice every week and the cells were split 1:4 or 1:8 every week.

Chemicals

Results and discussion

As a standard ROS for the DCF assay, we first tested H2O2. Figure 1 shows the concentration-response relationship of cells exposed to H2O2. Similar to the results found by LeBel et al. [4], the percentage increase of fluorescence is linearly correlated (r = 0.992) with the concentration of H2O2 in the range of 50 μM to 1 mM. A similar linearly correlated (r = 0.993) concentration-response relationship was found when using AAPH as the free radical generator in the same concentration range (Fig.

Acknowledgements

We thank Dr. Gerard Dallal for his advice in statistical analysis.

References (22)

  • D.A. Bass et al.

    Flow cytometry studies of oxidative product formation by neutrophilsa graded response to membrane stimulation

    J. Immunol.

    (1982)
  • Cited by (0)

    1

    Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.

    View full text