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Concerns in the application of fluorescent

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measure reactive oxygen species in vitro

Article in Toxicology in Vitro August 2015

DOI: 10.1016/j.tiv.2015.08.010 Source: PubMed

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 Toxicology in Vitro 30 (2015) 578582

Contents lists available at ScienceDirect

Toxicology in Vitro

journal homepage: www.elsevier.com/locate/tiv

Review

Concerns in the application of uorescent probes DCDHF-DA, DHR 123

and DHE to measure reactive oxygen species in vitro
Mazyar Yazdani
Department of Biosciences, University of Oslo, P.O. Box 1066, Blindern, N-0316 Oslo, Norway

a r t i c l e i n f o a b s t r a c t

Article history: Reactive oxygen species (ROS) are formed in biological systems by partial reduction of molecular oxygen. The
Received 3 July 2015 essential role of ROS in maintaining physiological health may be corrupted into oxidative stress by their over-
Received in revised form 30 July 2015 production or the exhaustion of antioxidant mechanisms. Many studies covering a broad range of methodologies
Accepted 18 August 2015
have investigated ROS production and their toxic mechanisms of action. Of these methodologies, uorometry
Available online 28 August 2015
has been among the preferred techniques. Three frequently used uorescent probes for in vitro studies
Keywords:
are 2,7-dichlorodihydrouorescein diacetate (DCDHF-DA), Dihydrorhodamine 123 (DHR 123) and
Reactive oxygen species (ROS) Dihydroethidium (DHE). Apart from the unavoidable limitations of auto-oxidation, photo-oxidation and
Oxidative stress photo-conversion, there are also concerns relating to protocol modication for the improved monitoring of
Fluorescence probes ROS. This paper aims to highlight such contributing factors, including cell culture conditions and the characteris-
DCDHF-DA tics of individual uorescent probes in the utilization of these selected probes in in vitro systems.
DHR 123 2015 Elsevier Ltd. All rights reserved.
DHE

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
2. Formation of uorescent products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
3. Cell culture conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
4. Fluorescent probe characteristics and inuencing factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581

1. Introduction Historically, the uorescent probes 2,7-dichlorodihydrouorescein

Reactive oxygen species (ROS) may be formed by partial reduction
of molecular oxygen through endogenous (physiological) or exogenous
(environmental) oxidative processes in biological systems. ROS repre-
sent a broad category of molecules consisting of both radical and non-
radical species. The former includes superoxide anion radical (O2),
hydroxyl radical (HO), peroxyl radical (RO2), alkoxyl radical (RO)
and hydroperoxyl radical (HO2). The non-radical species include
hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxygen
and peroxynitrite (ONOO). ONOO can also be categorized as a reactive
nitrogen species (RNS) (Gomes et al., 2005; Halliwell and Gutteridge,
2007; Sorg, 2004).
E-mail address: mazyar.yazdani.edu@gmail.com.
diacetate (DCDHF-DA), Dihydrorhodamine 123 (DHR 123) and
Dihydroethidium (Hydroethidine/HET; DHE) were often employed by
researchers for monitoring the formation of ROS in cells. Keston and
Brandt in 1965 were the rst to use DCDHF-DA for measuring hydrogen
peroxide uorometricly in the presence of horseradish peroxidase
(HRP) (Crow, 1997; Keston and Brandt, 1965). Subsequently, it became
a widely used uorophore to detect intracellular ROS in various cell-
types. To the confusion of many researchers, DCDHF-DA has been re-
ferred to by many names including DCDHF-DA, DCFH-DA, DCHF-DA,
DCFH2-DA and H2DCFDA (Kweon et al., 2001; Chen et al., 2010).
A chloromethyl derivative of this probe, known as CM-H2DCFD, has
been developed for more precise monitoring of intracellular ROS as
DCDHF-DA tends to leak out after diffusing into cells (Haugland, 1996;
Kweon et al., 2001). DHR 123 was rst presented in the late 1980s. It
is an analog of DCDHF-DA and differs to that compound in its lack of

http://dx.doi.org/10.1016/j.tiv.2015.08.010
0887-2333/ 2015 Elsevier Ltd. All rights reserved.
 M. Yazdani / Toxicology in Vitro 30 (2015) 578582
diacetate (DA) and dichloro substituents of DCDHF-DA. In addition, the
two hydroxyl groups of DCDHF-DA have been replaced by amino groups
(Crow, 1997). DHE was rst used by Gallop et al. (1984) as a redox
probe. The ability of DHE to freely permeate cell membranes has result-
ed its wide use for monitoring ROS (Gomes et al., 2005).
Unavoidable limitations such as auto-oxidation, photo-oxidation,
photo-bleaching, photo-conversion, cytotoxicity and non-specicity
may interfere with the formation of uorescence and thus the detection
of ROS (Chen et al., 2010; Gomes et al., 2005). Nevertheless, better
results should be possible if technical concerns in their application are
taken into account. The aim of this review is to summarize some of
the contributing factors, including cell culture conditions and uores-
cent probe characteristics in the utilization of DCDHF-DA or its deriva-
tives (so-called uorescein-based probes), DHR 123 and DHE in
in vitro systems.
2. Formation of uorescent products
Theoretically, a photon of excitation light is absorbed by an electron
of a uorescent molecule, a so-called uorophore, raising the energy
level of the electron to an excited state. This excitation period is short
and the electron returns to the ground state through the dissipation of
excitation energy via molecular collisions or transference to a proximal
molecule, and then the emission of the remaining energy as a photon
(Jameson et al., 2003).
DCDHF-DA, a membrane permeable dye, passively diffuses into
cells, where its acetate groups are actively cleaved by esterases to
form 2,7-dichlorodihydrouorescein (DCDHF). It is then oxidized by
free radical compounds to the uorescent 2,7-dichlorouorescein
(DCF) (Hempel et al., 1999). DHR 123, a lipophilic non-uorescent
molecule, readily diffuses across cell membranes and is efciently oxi-
dized by free radicals to form a uorescent cationic and lipophilic
probe rhodamine 123 (Rh 123) (Gomes et al., 2005; Kweon et al.,
2001). In the case of DHE, the probe undergoes a two-electron oxidation
by free radicals inside the cell resulting in the formation of ethidium
(E+), a uorescent compound (Fig. 1). It is believed that its uorescence
increases after binding to DNA, resulting in red uorescence (Gomes
et al., 2005).
3. Cell culture conditions
Cell numbers vary from experiment to experiment and this can in-
uence uorometer-based assays such as oxidative stress evaluation.
Cell culture densities may also uctuate as a result either natural cell
death or cell death induced by stressors, so researchers should be mind-
ful of this when measuring ROS. There are some contradictory ndings
regarding links between these two parameters (Maeda et al., 1993;
Saeki et al., 1997; Yasaka et al., 1996; Yasui et al., 2000). In one example,
Long et al. (2003) challenged the role of oxidative stress and apoptosis
in three different densities of mouse broblast cell lines. The results of
their study revealed that spontaneous apoptosis increased with increas-
ing cell-density, whereas H2O2-induced apoptosis was greater at lower
densities. In contrast, Li et al. (1999) reported a linearly increasing
rate of HO production with increasing cell density. The authors investi-
gated their hypothesis in a series of mouse epidermal cell lines exposed
to the anti-cancer compound diaziquone (AZQ). Such a controversy
has been linked to the use of different cell types (Long et al., 2003).
The expression of ROS formation in connection with cell density can
therefore be obtained by normalizing the measured ROS on the number
of viable cells (Bopp et al., 2008). In addition, a preliminary study
to determine the optimal cell number with regard to density-
dependence of cell death and degree of yielded uorescence linked
to oxidative stress would be useful.
Fluorescent probes have been used to detect ROS in both basic
systems for growing cells in suspension (non-adherent) and adherent
cultures (Hempel et al., 1999; Misra and Niyogi, 2009; Oya et al.,
579
1986; Pourahmad and O'Brien, 2000). The free-oating cells in suspen-
sion culture facilitate the immediate application of probes; while cells in
adherent culture require an incubation time ranging from a few hours
up to a day. This time gap allows the seeded cells to recover and attach
to coated cell/tissue culture plates with an articial substrate such as
polyornithine, laminin, Poly-L-lysine, bronectin or matrigel (Hempel
et al., 1999; Hynes et al., 2006; Long et al., 2003; Nat et al., 2007;
Nawaz et al., 2005b; Radice et al., 2004). This time interval in adherent
cultures also helps the cells to achieve some degree of equilibrium
with the in vitro condition (Dickson, 1971). For example, time series
studies evaluating some cellular parameters in adherent primary
culture of trout hepatocytes have shown changes (Ferraris et al., 2002;
Yazdani et al., 2015). In addition, the resultant suspension culture
from tissue perfusion procedure appeared to be a mixture of cell types
including red blood cells (Bonney et al., 1974; Dickson, 1971). For
removing red blood cells, nonadherent tissue cells and debris, changing
the medium or washing the culture with buffers has been suggested
(Hempel et al., 1999; Manzl et al., 2004).
4. Fluorescent probe characteristics and inuencing factors
DCDHF-DA and its derivatives, DHR 123 and DHE have routinely
been used in in vitro studies and some authors claim that they specical-
ly detect intracellular hydrogen peroxide, peroxynitrite and superoxide,
respectively (Bindokas et al., 1996; Bindokas et al., 2003; Kooy et al.,
1994; Rico et al., 2009; Roy et al., 2009). In contrast, the non-
specicity of some uorescent probes including DCDHF-DA (HO,
H2O2, RO2), DHR 123 (H2O2/HRP, HOCl) and DHE (O2, H2O2, ONOO,
HOCl) has been well reviewed in e.g. Gomes et al. (2005), Tarpey and
Fridovich (2001) and Wardman (2007).
In the uorescent labeling procedure, one must initially determine
the concentration-response relationship between the probe and the
uorescent signal (Braut-Boucher and Aubery, 2010). Manufacturers'
protocols (e.g. Invitrogen Life Technologies) have suggested an empiri-
cal determined nal working concentration of dyes of ~ 110 M in a
simple physiological buffer such as phosphate buffered saline (PBS),
Hank's buffered saline solution (HBSS) and 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) rather than a complex media
for cell culture. In order to gain a stronger uorescent signal, the
application of minimal loading dose is recommended. The intracellular
environment can be disturbed at high concentrations because the
probes can work as either antioxidants or prooxidants. In addition, leak-
age and quenching can also occur at higher probe concentrations
(Braut-Boucher and Aubery, 2010; Zuo and Clanton, 2002). Royall and
Ischiropoulos (1993) reported N 90% loss of intracellular probes in endo-
thelial cell cultures loaded with 11 M DCDHF-DA, 11 M DCF or 5 M
DHR 123 for 1 h, followed by 1 hour exposure to probe-free medium.
In the same study, intracellular Rh 123 decreased by only 15%. High
concentrations of DHE have been linked with the formation of
superoxide-independent uorescence via saturation of mitochondrial
nucleic acid binding sites by derived E+, facilitating its bonding to nucle-
ar DNA (Budd et al., 1997).
Fluorescent probes are readily available as premade solutions,
but some researchers may prefer to use the powder forms. In these
cases, the choice of solvent requires the careful consideration of its
properties. Water is poor solvent of acetylated dye powders and thus
requires the initial use of high-grade solvents such as dimethyl sulfoxide
(DMSO), dimethylform-amide (DMF) or 100% ethanol prior to empiri-
cal work. The dissolved DCDHF-DA and DHR 123 are transparent solu-
tions which, after purging with N2, is stored in the dark at 20 C to
prevent auto-oxidation. Contrastingly, a freshly prepared DHE uores-
cent probe possesses a light pink color, which darkens as it ages, even-
tually turning red due to auto-oxidation. This can therefore affect the
sensitivity of assay through increased background uorescence (Zuo
and Clanton, 2002).
580 M. Yazdani / Toxicology in Vitro 30 (2015) 578582

Fig. 1. Chemical structure and oxidation of 2,7-dichlorodihydrouorescein diacetate (DCDHF-DA), Dihydrorhodamine 123 (DHR 123) and Dihydroethidium (DHE).

The cells are loaded with uorescent probes for 260 min either
before (preloading) or after (post-loading) adding exposure medium
(Bopp et al., 2008; Guerra et al., 2005; Mathisen et al., 2007; Misra
and Niyogi, 2009; Nawaz et al., 2005a; Pourahmad and O'Brien, 2000;
Rico et al., 2009; Winzer et al., 2000). These pre- or post-loading proce-
dures may cause confusion since there is no general agreement on their
application. Preloaded cells are more likely to receive a uniform dose of
the probes through their intact membranes; post-loading of already
stressed cells may result in an uneven uptake of the probes, as well
as the rapid leakage of them. In addition, decreased enzyme activity
in latter case can inuence the measurement of ROS in enzyme-
dependent probes (Joux and Lebaron, 2000). Nonetheless, the post-
loading procedure may be considered the preferred procedure since
retention of the probes within the cell is time dependent due to the
effects of passive diffusion and active efux. The compartmentalization
of probes in cells and the cytotoxic effect of their metabolites may also
favor the latter procedure (Chen et al., 2010; Joux and Lebaron, 2000;
Wardman, 2007).
The loading temperature is one of the key issues in enzyme depen-
dent uorescent probes. In the case of DCDHF-DA and DHR 123, esterase
activity is signicantly affected by temperature during cleavage. Never-
theless, this step should be carried out at the optimal cell growth
temperature so as to avoid any stress (e.g. oxidative stress) resulting
from temperature shock. For example, Farmen et al. (2010) used room
temperature for loading CM-H2DCFDA to the primary cultures of rain-
bow trout hepatocytes. The optimal incubation temperature for this
cell culture is 15 C (Braunbeck and Neumuller, 1998; Schreer et al.,
2005; Tollefsen et al., 2003; Yanhong et al., 2008) and exposed trout
 M. Yazdani / Toxicology in Vitro 30 (2015) 578582
hepatocytes to heat shock (+15 C for 1 h) indicated higher accumula-
tion of hsp70 over a 24 h period (Boone and Vijayan, 2002). One
solution may be the extension of loading times for cells which temper-
atures lower than those recommended for loading of specic probes.
Loading times for DHE seem to be largely independent of temperature
(Zuo and Clanton, 2002).
The quantication of free radicals using uorescent probes is often
sensitive to pH. Kalinich et al. (1997) showed the effect of pH on
Trolox-induced DCDHF-DA oxidation. Trolox, a water-soluble vitamin
E analog, caused very little formation of DCF uorescence at pH less
than 7.0 in their study while remarkable increase occurred at pH values
above 7.0. Kooy et al. (1994) studied the effect of pH (4.210.3) on
peroxynitrite-mediated oxidation of DHR 123 and found a constant
oxidation between pH 4.2 and 8.3. They also reported a signicant
decrease at pH N 8.3.
5. Conclusion
There has been a growing interest in the use of uorescent probes
to monitor ROS in in vitro systems as they are often seen to be the
preferred technique and excellent sensors for ROS detection. In order
to obtain more empirically robust results, it is crucial to consider modi-
cations to protocols under experimental conditions. Contributing
factors such as cell culture conditions and the characteristics of individ-
ual uorescent probes must also be considered and careful adjustment
should be weighted for each model and each probe prior to the running
of experiments.
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