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What does the commonly used DCF-test for oxidative
stress really show?
Markus Karlsson, Tino Kurz, Ulf T Brunk, Sven E Nilsson, Christina I
Frennesson
To cite this version:
Markus Karlsson, Tino Kurz, Ulf T Brunk, Sven E Nilsson, Christina I Frennesson. What does the
commonly used DCF-test for oxidative stress really show?. Biochemical Journal, Portland Press, 2010,
428 (2), pp.183-190. �10.1042/BJ20100208�. �hal-00483251�
What does the commonly used DCF-test for oxidative stress really show?
Markus KARLSSON*, Tino KURZ†, Ulf T. BRUNK†, Sven E. NILSSON*, Christina I.
FRENNESSON*
*Division of Ophthalmology, Faculty of Health Sciences, Linköping University, 581 85 Linköping,
SWEDEN and †Division of Pharmacology, Faculty of Health Sciences, Linköping University, 581
85 Linköping, SWEDEN
Address correspondence to: Ulf T. Brunk, Division of Pharmacology, Faculty of Health Sciences,
Linköping University, 581 85 Linköping, SWEDEN.
Tel.: +46-13-221515
Fax: +46-13-149106
E-mail: ulf.brunk@liu.se
SYNOPSIS
Dihydrodichlorofluorescein (H
2
DCF-DA) is widely used to evaluate “cellular oxidative stress”.
After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-
esterified to a hydrophilic alcohol (H
2
DCF) that may be oxidized to fluorescent DCF by a process
usually considered to involve reactive oxygen species (ROS). It is, however, not always recognized
that, being a hydrophilic molecule, H
2
DCF does not pass membranes, except for the outer,
fenestrated mitochondrial ones. It is also not generally realized that oxidation of H
2
DCF is
dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c,
for neither superoxide, nor hydrogen peroxide, directly oxidizes H
2
DCF. Consequently, oxidation
of H
2
DCF requires the presence of either cytochrome c or of both redox-active transition metals and
hydrogen peroxide. Redox-active metals exist mainly within lysosomes, while cytochrome c resides
bound to the outer side of the inner mitochondrial membrane. Following exposure to H
2
DCF-DA,
weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the
much more sensitive J774 cells. This fluorescence was only marginally enhanced following short
exposure to hydrogen peroxide, showing it by itself being unable to oxidize H
2
DCF. Cells that were
either exposed to the lysosomotropic detergent MSDH, exposed to prolonged oxidative stress, or
spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence.
The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of
lysosomal iron and/or mitochondrial cytochrome c.
SHORT TITLE: DCF and oxidative stress
KEYWORDS: DCF, lysosomes, oxidative stress, ROS, transition metals.
ABBREVIATIONS
AMD, age-related macular degeneration; AO, acridine orange; CP22, 1-propyl-2-methyl-3-
hydroxypyrid-4-one; DCF, dichlorofluorescein; DMEM, Dulbecco’s Modified Eagle’s Medium;
DMSO, dimethyl sulfoxide; FAC, ferric ammonium citrate; FBS, fetal bovine serum; H
2
DCF,
dihydrodichlorofluorescein; H
2
DCF-DA, dihydrodichlorofluorescein diacetate; H
2
O
2
, hydrogen
peroxide; HBSS, Hank’s buffered salt solution; LMP, lysosomal membrane permeabilization;
MMP, mitochondrial membrane permeabilization; MSDH, O-methyl-serine dodecylamide
hydrochloride; ROS, reactive oxygen species; TMRE, tetramethylrhodamine ethyl ester.
Biochemical Journal Immediate Publication. Published on 23 Mar 2010 as manuscript BJ20100208
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20100208
Accepted Manuscript
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
© 2010 The Authors Journal compilation © 2010 Portland Press Limited
DCF and oxidative stress
2
INTRODUCTION
The molecular mechanisms behind age-related macular degeneration (AMD) are much debated
but far from understood, even though it is the most common cause of visual impairment among the
elderly in the western world [1-3]. However, based on recent evidence, oxidative stress within
retinal pigment epithelial (RPE) cells, with the ensuing accumulation of lysosomal lipofuscin and
thereby depressed phagocytosis/autophagocytosis, is considered a major etiological factor behind
AMD [4-8]. Consequently, in relation to various experimental studies on cultures of RPE cells, the
extent of cellular oxidative stress is often being evaluated.
A method that is frequently relied upon for this purpose is the DCF-test [9-11].
Dihydrodichlorofluorescein diacetate (H
2
DCF-DA) is then added to cells in culture and the
intracellular oxidation of H
2
DCF to DCF documented over time. H
2
DCF-DA is a non-fluorescent
lipophilic ester that easily crosses the plasma membrane and passes into the cytosol, where it is
rapidly cleaved by unspecific esterases [12]. One of the reaction products is the non-fluorescent
alcohol H
2
DCF. The oxidation of this molecule to the fluorochrome DCF results in green
fluorescence when excited with blue light. The brightness of this fluorescence is usually considered
to reflect the extent to which ‘reactive oxygen species’ (ROS) are present, unfortunately without
any further definition of what kind of ROS may give rise to the oxidation [13, 14]. Furthermore, it
is often wrongly assumed that H
2
DCF is evenly distributed in the cell following the intracellular
cleavage of the added diacetate ester, neglecting the inability of hydrophilic molecules to traverse
membranes. Moreover, the evaluation of the DCF-test commonly involves plate readers or flow
cytofluorometers, which do not allow any careful morphological analysis of individual cells that, if
undertaken, might have disclosed unexpected cellular variations. Thus, the DCF-test often seems to
be somewhat uncritically used.
We previously showed that oxidation of H
2
DCF to DCF is not a result of exposure to reactive
oxygen species (ROS) in general, but rather indicates the specific impact of hydroxyl radicals
formed during Fenton-type reactions [15]. Here we also stress that cytochrome c, as has been
previously pointed out [16-18], operates as an unspecific peroxidase with H
2
DCF as a target. It is,
therefore, plausible to suppose that lysosomal membrane permeabilization (LMP), with release to
the cytosol of redox-active iron, and/or mitochondrial release of cytochrome c is required for the
induction of strong cytosolic DCF-mediated fluorescence. We also point out that in the absence of
LMP and apoptosis/necrosis, H
2
DCF only occurs in the cytosol and the mitochondrial inter-
membranous space, where it gives rise to weak cytosolic and somewhat stronger mitochondrial
fluorescence that indicates oxidation. Most probably, this faint fluorescence, that regularly seems to
be considered background and to be over-looked, results from the normal mitochondrial production
of hydrogen peroxide that diffuses all over the cell, the presence of cytochrome c in the
mitochondrial inter-membranous space as well as of minute amounts of labile iron under transport
in the cytosol. Consequently, if lysosomal membrane permeabilization (LMP) and related
mitochondrial damage with relocation of cytochrome c has taken place, a positive DCF-test might
be interpreted as a sign of oxidative stress even if that would not necessarily be the case. Similarly,
a low or undetectable level of DCF fluorescence may not necessarily indicate the absence of ROS
but rather designate stable lysosomal and mitochondrial compartments.
MATERIALS AND METHODS
Chemicals
Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s F12 medium, fetal bovine serum (FBS),
penicillin and streptomycin were from Invitrogen (Paisley, UK). Acridine orange base (AO) was
from Gurr (Poole, UK), 1-propyl-2-methyl-3-hydroxypyrid-4-one (CP22) and O-methyl-serine
dodecylamide hydrochloride (MSDH) were kind gifts from Prof. Robert Hider, University of
Biochemical Journal Immediate Publication. Published on 23 Mar 2010 as manuscript BJ20100208
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20100208
Accepted Manuscript
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
© 2010 The Authors Journal compilation © 2010 Portland Press Limited
DCF and oxidative stress
3
London, (UK) and Dr. Gene N. Dubowchik (Wallingford, CT, USA), respectively. All other
reagents were from Sigma (St. Louis, MO, USA).
Cells and culture conditions
ARPE-19 (human immortalized retinal pigment epithelial) cells and murine J774 macrophage-
like histiocytic lymphoma cells (both obtained from ATCC, Manassas, VA, USA) were grown at
37°C in humidified air with 5% CO
2
in DMEM and F12 (1:1) supplemented with 10% FBS, 2 mM
L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were sub-cultivated twice a
week, seeded in 6-well plates with or without cover slips at a concentration of 5x10
5
cells/well
(J774), while the much larger ARPE-19 cells were seeded at 2x10
5
cells/well with or without cover
slips. Cells were subjected to experiments 12, 24 and 48 h following subcultivation.
Conditions for basic studies with H
2
DCF-DA only
Small amounts of stock solutions, containing 1 to 10 mM H
2
DCF-DA in DMSO, were added to
fresh complete growth medium. The carefully blended solution was added to the culture dishes
following removal of the old medium. Cells on cover slips were in that way exposed to 3-30 µM
H
2
DCF-DA in medium with 0.03-3% DMSO for 30 min at otherwise standard culture conditions.
The cells were then rinsed in HBSS, directly mounted in HBSS, and within 5-6 min subjected to
laser scanning confocal microscopy using a Nikon Eclipse C1 laser scanning confocal microscope
(Tokyo, Japan).
Following tests with varying concentrations of H
2
DCF-DA and DMSO, a stock solution of 10
mM H
2
DCF-DA in DMSO was selected for both cell types and added to medium in such a way that
the final concentrations of H
2
DCF-DA and DMSO was 10 µM and 0.1%, respectively. The cells
were observed and photographed using the above confocal microscope, r and the Nikon EZ-C1
V3.70 software for image acquisition. DCF fluorescence was detected using a 515/30 nm band pass
filter. Since the DCF fluorescence of control cells is rather weak, the medium size pinhole and an
electronic gain of 6.5 were applied. All photographs were taken at a resolution of 2 Mpixels. To
allow comparison between experiments, these settings were then kept the same for all experiments
involving evaluation of DCF-induced fluorescence, even when the intensity of fluorescence was
much enhanced due to the conditions being applied and, consequently, a small pinhole and less
amplification would have given better resolution and prevented over-exposure of strongly
fluorescent cells.
Localisation of mitochondria using the mitochondria-specific dye TMRE
Mitochondria were demonstrated using the cationic and lipophilic dye tetramethylrhodamine
ethyl ester (TMRE), which accumulates in the matrix of normal mitochondria. Cells were incubated
with TMRE in complete culture medium (100 nM; 15 min; 37°C) and observed using the above-
mentioned Nikon confocal microscope. Since mitochondrial TMRE fluorescence is strong, optimal
documentation conditions were used (the smallest pinhole and a low gain).
Overlays of DCF/TMRE pictures could not be produced because TMRE is a metachromatic
fluorophore that provides both red and green fluorescence when activated by a 488 nm laser and the
green one is much stronger than that of DCF in control cells. TMRE fluorescence was detected
using a 590/50 nm band pass filter.
Studies aiming at varying cytosolic labile iron concentrations before exposure to H
2
DCF-DA
To evaluate the influence on DCF-induced fluorescence of different concentrations of labile iron
in the cytosol, as well as in the mitochondrial inter-membranous space, cells were initially exposed
to H
2
DCF-DA as above and then mounted in HBSS with 500 µM ferric ammonium citrate (FAC) to
enhance labile iron. Separately, cells were incubated with H
2
DCF-DA together with 100 µM of the
iron chelator 1-propyl-2-methyl-3-hydroxypyrid-4-one (CP22) to ligate labile iron and then mounted
Biochemical Journal Immediate Publication. Published on 23 Mar 2010 as manuscript BJ20100208
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20100208
Accepted Manuscript
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
© 2010 The Authors Journal compilation © 2010 Portland Press Limited
DCF and oxidative stress
4
in HBSS in the continuous presence of CP22. CP22 (MW 203.7) is an effective, water-soluble
bidentate iron chelator with a complex constant of a10
36
[19]. When complexed with CP22, iron is
prevented from redox-cycling and unable to support Fenton-type reactions.
Studies aiming at evaluating DCF-fluorescence in relation to oxidative stress
1. Exposure to hydrogen peroxide. In order to induce oxidative stress, ARPE-19 and J774 cells
were initially exposed to H
2
DCF-DA as above and then mounted in HBSS with 500 µM H
2
O
2
.
To induce apoptosis of the ARPE-19 (very resistant to oxidative stress) and J774 (sensitive) cells,
they were exposed to 15 mM and 100 µM H
2
O
2
in HBSS, respectively, for 30 min and then kept at
standard culture conditions for about 6 h when they were incubated with H
2
DCF-DA and studied as
described above. In order to further emphasize the differences in lysosomal membrane stability
between cell types, both of them were exposed to 100 µM H
2
O
2
in HBSS for 30 min and then kept
at standard culture conditions for about 6 h, when they were exposed to the lysosomotropic,
metachromatic fluorochrome acridine orange (AO) as described before [20-24]. A 488 nm argon
laser was used for excitation and AO fluorescence was detected using 515/30 nm (green) and
590/50 nm (red) band pass filters.
2. Exposure to a lysosomotropic detergent. In order to induce lysosomal rupture by other means
than oxidative stress, ARPE-19 cells were initially exposed to H
2
DCF-DA as above. They were
then mounted in HBSS with 100 µM of the lysosomotropic detergent O-methyl-serine
dodecylamide hydrochloride (MSDH), which is known to induce lysosomal labilization [25], before
being studied over a 15 min period of time. In separate experiments, designed to verify the LMP-
effect of MSDH, cells were initially exposed to AO as above and then mounted in HBSS with
MSDH and followed over time.
Evaluation of DCF-induced fluorescence in apoptotic ARPE-19 cells
Apoptotic and post-apoptotic necrotic cells are always present in cell cultures, especially shortly
after subcultivation, because a number of cells do not survive that procedure. Such apoptotic ARPE-
19 cells were identified, 12 h following seeding, based on their characteristic morphology that
includes chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and formation
of apoptotic bodies. DCF-induced fluorescence was evaluated following exposure to H
2
DCF-DA as
described above.
Assessment of the capacity of cytochrome c to oxidize H
2
DCF
Using a modification [15] of a technique described by Myhre et al. [26], H
2
DCF was exposed to
cytochrome c +/- the small iron chelator CP22 (at 1 and 20 µM, respectively). Briefly, a standard
consisting of ferric iron (10 µM) was reduced to its ferrous form by cysteine (100 µM) in a HEPES
buffer (pH 7.0; 150 mM). Hydrogen peroxide (100 µM) was added to initiate oxidative conversion
of non-fluorescent H
2
DCF (5 µM) to fluorescent DCF [H
2
DCF was obtained by hydrolyzing its
diacetate ester (H
2
DCF-DA)]. DMSO (10%) and CP22 (20 µM) were used to demonstrate the
involvement of HO
•
and iron, respectively.
The capacity of cytochrome c to oxidize H
2
DCF was then compared to that of the Fe(II) standard.
Fluorescence was measured in a FL600 Microplate Fluorescence Reader (Bio-Tek, Winooski, VT,
USA) at Ȝ
ex
485 nm and Ȝ
em
530 nm.
Statistics
DCF-induced fluorescence intensity was measured using the NIH ImageJ software v. 1.42q.
Following delineation of each cell, their mean fluorescence was calculated and analysis performed
using Student’s t-test.
RESULTS
Biochemical Journal Immediate Publication. Published on 23 Mar 2010 as manuscript BJ20100208
THIS IS NOT THE VERSION OF RECORD - see doi:10.1042/BJ20100208
Accepted Manuscript
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.
© 2010 The Authors Journal compilation © 2010 Portland Press Limited
