In this study, a fast-reaction fluorescent probe for O2Æ, 2-chloro-1,3-dibenzothiazolinecyclohexene DBZTC; Scheme 1, was synthesized in-house and characterized using 1H NMR, IR spectra an
Trang 1from macrophages by using a novel fluorescent probe
Jing Jing Gao1, Ke Hua Xu1, Bo Tang1, Ling Ling Yin1, Gui Wen Yang2and Li Guo An2
1 College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan China
2 College of Life Science, Shandong Normal University, Jinan, China
Reactive oxygen species (ROS) such as the superoxide
anion radical (O2Æ), hydroxyl radical (HOÆ) and
hydro-gen peroxide (H2O2) are important mediators in
various pathological diseases [1] Free radicals, in
par-ticular O2Æ, have been found extensively in myocardial
ischemia and reperfusion injury in recent years [2]
Excessive O2Æ, a toxic reactive oxygen, not only harms
many biological molecules, but can also be converted
to other more toxic radicals such as HOÆ, H2O2, 1O2,
and so on [3] Under consenescence or special physical
conditions, excessive O2 Æ can lead to coronary
arterio-sclerosis and tumors [4–6] Therefore, real-time
monit-oring of O2Æ under physiological conditions is of
increasing importance
To date, methods to detect of O2 Æ include ESR [7],
superoxide dismutase (SOD)-inhibitable Nitro Blue
tetrazolium [8], chemiluminescence [9,10] and
fluor-escence [11] Among these methods, fluorescence
detection appears to be particularly attractive because
it is able make superoxide anion radicals in living cells
‘visible’ in situ using confocal laser scanning micro-scopy Although several types of fluorescent probe for detecting and imaging O2 Æ have been described [11– 13], those with high selectivity, sensitivity and practi-cality are rare For example, hydroethidine is the most commonly used fluorescent probe for O2 Æ [14], but its selectivity could be improved [15] In addition, owing
to the short half-life of the superoxide anion radical, there is an exigent need for researchers to develop fast-response probes to trap O2 Æ in order to investigate its mode of production, metabolism and trafficking Therefore, considering the design-applicable probes, it
is important that the reaction rate and selectivity are improved to avoid potential side reactions from other ROS under conditions that guarantee high sensiti-vity If novel fluorescence probes that overcome these
Keywords
2-chloro-1, 3-dibenzothiazolinecyclohexene;
fluorescence image; fluorescent probe;
macrophages; superoxide anion radical
Correspondence
B Tang, College of Chemistry, Chemical
Engineering and Materials Science,
Engineering Research Center of Pesticide
and Medicine Intermediate Clean
Production, Ministry of Education, Shandong
Normal University, Jinan 250014, China
Fax: +86 531 861 80017
Tel: +86 531 861 80010
E-mail: tangb@sdnu.edu.cn
(Received 24 October 2006, revised 11
January 2007, accepted 29 January 2007)
doi:10.1111/j.1742-4658.2007.05720.x
Quantitation of superoxide radical (O2Æ) production at the site of radical generation remains challenging A simple method to detect nanomolar to micromolar levels of superoxide radical in aqueous solution has been devel-oped and optimized This method is based on the efficient trapping of O2 Æ using a novel fluorescent probe (2-chloro-1,3-dibenzothiazolinecyclohex-ene), coupled with a spectra character-signaling increase event A high-spe-cificity and high-sensitivity fluorescent probe was synthesized in-house and used to image O2 Æ in living cells Better selectivity for O2 Æ over competing cellular reactive oxygen species and some biological compounds illustrates the advantages of our method Under optimal conditions, the linear calib-ration range for superoxide anion radicals was 5.03· 10)9)3.33 · 10)6m The detection limit was 1.68· 10)9m Fluorescence images of probe-stained macrophages stimulated with 4b-phorbol 12-myristate 13-acetate were obtained successfully using a confocal laser scanning microscope
Abbreviations
DBZTC, 2-chloro-1,3-dibenzothiazolinecyclohexene; ROS, reactive oxygen species; SOD, superoxide dismutase.
Trang 2problems were available, they would contribute greatly
to the elucidation of the roles of O2 Æ in living cells
In this study, a fast-reaction fluorescent probe for
O2Æ, 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC;
Scheme 1), was synthesized in-house and characterized
using 1H NMR, IR spectra and elemental analysis In
an experiment, a xanthine⁄ xanthine oxidase (XA ⁄ XO)
model system provided sustained O2 Æ production
DBZTC was able to react with O2 Æ and yield a strong
fluorescence product with an emission maximum at
559 nm Reaction of DBZTC with O2 Æ was
accom-plished within 10 min, making it more practical than
the probes reported previously [12] for detecting O2Æ
directly in living cells More promisingly, the proposed
method has better selectivity for O2 Æ over other ROS
and biological compounds, especially H2O2 Because
the level of H2O2 is high compared with O2 Æ, due to
accumulation in biological systems, H2O2 may be the
most competitive ROS in the quantitative detection of
O2Æ In this study, DBZTC showed a selective
response to O2Æ that was > 500-fold greater than the
response to H2O2 (ratio in mol), which provides a
unique opportunity to develop a chemical tool to
mon-itor O2 Æ in a specific manner Under optimal
condi-tions, a linear relationship between relative
fluorescence intensity and O2Æ concentration was
obtained in the range 5.03· 10)9)3.33 · 10)6m The
detection limit was 1.68· 10)9m In this regard,
DBZTC is well suited as a fluorescent reagent that
allows the cellular chemistry of O2Æ to be examined at
the molecular level
Results and Discussion
Spectral properties
Initially, we investigated the spectral properties of
the probe under simulated physiological conditions
(Hepes, 0.02 m, pH 7.4) As indicated in Fig 1, DBZTC showed low blank fluorescence, although the addition of different concentrations of XA⁄ XO trig-gered prompt increases in fluorescence (kex ⁄ em¼
485⁄ 559 nm) The XA ⁄ XO system was used as the main source of O2 It has been reported that on the catalysis of XO at pH 7.40, XA is oxidized to uric acid [16] XA + 2O2+ H2O¼ uric acid + 2O2Æ + 2H+ (single electron transfer) XA can also be oxidized by
O2 with double electrons XA + O2+ H2O¼ uric acid + H2O2 (double electron transfer) During the process, five of the six electron transfers are double electron transfers, and one electron transfer is single Namely, one unit of XO can catalyze the conversion
of 1.00 · 10)6mol XA into 0.33· 10)6mol O2 Æ [17]
As expected, the fluorescence intensity increased with increasing O2Æ concentration (Fig 1) Moreover, there was a good linear correlation (R¼ 0.9941)
Scheme 1 Synthesis of DBZTC.
Fig 1 Emission spectra (kex¼ 485 nm) of DBZTC (10 l M ) in the presence of various concentrations of O 2–Æ (0–6.66 l M ) at 37 C in Hepes buffer (pH 7.40) Spectra were obtained 10 min after the addition of different concentrations XA ⁄ XO (final concentration:
0 ⁄ 0, 2 ⁄ 2, 3 ⁄ 3, 4 ⁄ 4, 6 ⁄ 6, 10 ⁄ 10, 15 ⁄ 15 and 20 ⁄ 20 l M ⁄ mU) to a solution of DBZTC.
Trang 3between fluorescent intensity and O2Æ concentration in
the range 5.03· 10)9)6.66 · 10)6m The regression
equation was F¼ 3815.8 [O2 Æ] (lm) +2579.1 (Fig 2)
The detection limit was 1.68· 10)9m This result
showed that DBZTC could detect O2Æ both
qualita-tively and quantitaqualita-tively
Reaction conditions
To determine the optimum reaction conditions for
the analysis of O2Æ, the effect of buffer solution and
the concentration of the fluorescent probe were
investigated
Effect of pH and buffer concentration
The pH of the medium has a large effect on
fluores-cence intensity Hepes was used because it has been
shown to be a better buffer for the incubation of
mammalian cells, and may improve the planting
efficiency of cell incubation and breed ability of cell
We showed that the optimal pH for O2Æ detection
was in the range 7.20–8.20 (Fig 3A) Buffer
concen-tration also affects fluorescence intensity We showed
that the relative fluorescence intensity of the system
was high and stable at buffer concentrations of 16–22
(v⁄ v %) (Fig 3B) Thus 20 (v ⁄ v %) of Hepes
(pH 7.40) was used throughout In fact, a lower
concentration of Hepes has a poor buffer capacity,
and superfluous Hepes leads to a salt effect that
decreases fluorescence intensity
Effect of the fluorescent probe concentration
The concentration of DBZTC directly decided whether
O2Æ was trapped completely, which determined the
precision and sensitivity of the analytical method The relative fluorescence intensity increased as the DBZTC concentration increased (< 9 lm), remained constant
at DBZTC concentrations of 9–18 lm, then decreased (Fig 4) A suitable concentration of DBZTC was advantageous, whereas superfluous DBZTC could quench fluorescence Therefore, 10 lm of DBZTC was used throughout
Effects of other ROS and biological compounds
To assess the selectivity of the method, the effect of other ROS and biological compounds on the determin-ation of 3.33 lm O2 Æ was examined individually An error of ± 5.0% in the relative fluorescence intensity was considered tolerable Little or no interference was encountered with (tolerance ratio in mol): NaClO, tert-butyl hydroperoxide, H2O2 (500); 1O2, glutathione, 1,4-hydroquinone (100); VC (50); HOÆ (5); ONOO–,
NO (1) O2 Æ was created by the enzymatic reaction of
Fig 2 A linear correlation between the fluorescence intensity and
O2– Æ concentration.
A
B
Fig 3 (A) Effect of pH (B) Effect of buffer concentration: DBZTC (10 l M ), XA (10 l M ), XO (10 mU), Hepes (20 m M ).
Trang 4XA⁄ XO (10 lm ⁄ 10 mU) at 25 C for 5 min HOÆ and
single oxygen (1O2) were generated by reacting H2O2
with Co2+ or NaOCl, respectively Peroxynitrite
(ONOO–) and nitric oxide (NOÆ) were obtained from
3-morpholinosydnonimine hydrochloride (SIN-1), and
3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene,
respectively The results are summarized in Fig 5
DBZTC appears to be a highly selective fluorescent
probe for O2Æ
Effect of SOD on fluorescence intensity of the reaction
of DBZTC with superoxide
In order to further confirm that the changes in fluores-cence of the probe solution were caused by O2 Æ, SOD,
a scavenger of O2 Æ, was used in the reactive system After reaction of SOD (150 U) with XA⁄ XO (10 lm⁄ 10 mU) in Hepes buffer (20 mm) had been car-ried out for 30 min at 37C, DBZTC was added and the reaction was immediately diluted with doubly dis-tilled water The mixture was equilibrated and kept for
5 min before measurement As can be seen from Fig 6, fluorescence intensity was markedly suppressed
by addition of SOD However, when SOD was replaced by heat-inactivated SOD (90C for 5 min) [18] or catalase, the fluorescence intensity of the reac-tion system barely changed Overall, the results sub-stantiated that the proposed method was effective for detecting O2 Æ
Reaction mechanism
We used XA⁄ XO as the source of O2Æ to simulate bio-logical systems In order to confirm the reaction mech-anism, IR and 1H NMR spectra of DBZTC and DBZTC oxide were analyzed As shown in the
1H NMR spectra of DBZTC oxide, peaks correspond-ing to N–H (4.5) and C–H (4.0) disappeared, and in the IR spectrum, N–H (3245 cm)1) and C–H (3110 cm)1) also disappeared, while a C¼ N absorp-tion band at 1618 cm)1 appeared All spectral data indicated that a larger conjugated structure of the DBZTC oxide was formed From product analysis and the fluorescence properties, we propose that the mode
Fig 4 Effect of the fluorescent probe concentration: XA (10 l M ),
XO (10 mU), Hepes (20 m M ).
Fig 5 Selectivity of DBZTC for O2–
Æ at pH 7.40 (20 m M Hepes).
Fluorescence response of DBZTC to O2– Æ, other ROS and biological
compounds Bars represent the final integrated fluorescence
response (F) over the initial integrated emission (F 0 ) Initial spectra
were acquired in a 10 l M solution of DBZTC Light grey bars
repre-sent the addition of an excess of the appropriate other ROS and
biological compounds (1.66 m M for H 2 O 2 , NaClO, and tert-butyl
hydroperoxide, 0.33 m M for 1 O2, GSH, and 1,4-hydroquinone,
0.17 m M for VC, 16.7 l M for HOÆ, 3.33 l M for ONOO – , NOÆ and
O 2–Æ) to a 10 l M solution of DBZTC Black bars represent the
sub-sequent addition of 3.33 l M O2–
Æ to the solution Excitation was provided at 485 nm.
Fig 6 Fluorescence response of 10 l M DBZTC to XA ⁄ XO (10 ⁄ 10 l M ⁄ mU) in the presence of SOD (150 U), heat-inactivataed SOD (150 U), catalase (150 U) or the absence of SOD, respectively (kex¼ 485 nm).
Trang 5of reaction of O2 Æ with DBZTC is as follows
(Scheme 2) Upon treatment with O2Æ, DBZTC, an
almost nonfluorescent compound, was oxidized at
pH 7.40 by deleting hydrogen [19] to yield DBZTC
oxide, which has better rigidity and a larger conjugated
system (free electron pairs on S also conjugating with
phenyl ring)
In order to validate the molar ratio of the reaction
between the probe and O2Æ, we synthesized pure
DBZTC oxide and tested the fluorescence intensity of
the oxide at different concentrations A linear
relation-ship between DBZTC oxide concentration and the
fluor-escence intensity was obtained (Fig 7) The linear
equation was as followed: F¼ 4268 [DBZTC oxide] +
288 Comparing this with the linear correlation between
the fluorescent intensity and the concentration of
O2Æ (F¼ 3815.8 [O2 Æ] + 2579.1), we may deduce that
DBZTC reacts with O2 Æ in a 1 : 1 molar ratio
Kinetic assay
Different reaction times were investigated XA⁄ XO
(10 lm⁄ 10 mU) was added to buffer solutions of
DBZTC (10 lm) maintained in a 37C water bath
The fluorescence emission of each reaction mixture was
measured at 5-min intervals against a reagent blank
using a fluorescence spectrometer Based on the
reac-tion mechanism of DBZTC towards O2Æ (1 : 1 molar ratio), the level of DBZTC was sufficient enough to capture O2Æ completely As shown in Fig 8, the fluo-rescence intensity of reaction system increased with increasing time up to 10 min and became constant thereafter This shows that the probe can capture O2 Æ quickly within 10 min, which attests to DBZTC being a
‘fast response’ probe The fluorescence of the reagent blank remained unchanged to 40 min, which indicates that the probe has good stability
Confocal fluorescence imaging and detection
of O2–Æ in cells
We used RAW264.7 macrophages to investigate the potential of DBZTC to detect O2 Æ in living cells Macr-ophages were seeded onto a glass slide and the concen-tration adjusted to 1· 106cellsÆmL)1 with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin Cells were loaded with DBZTC (10 lm, Hepes, pH 7.40) by incubation at 37C for 15 min and showed negligible intracellular background fluorescence (Fig 9A) Macr-ophages, which were loaded with DBZTC (10 lm), were stimulated with 4b-phorbol 12 myristate 13 acet-ate (2 ngÆmL)1) at 37C for 12 h; an obvious increase
Fig 7 Linear correlation between the fluorescence intensity and
the concentration of DBZTC oxide.
Scheme 2 A mechanism for the reaction of DBZTC with O2– Æ.
Fig 8 Effect of reaction time 1, DBZTC (10 l M ) + Hepes (20 m M ) + XA (10 l M ) + XO (10 mU); 2, DBZTC (10 l M ) + Hepes (20 m M ).
Trang 6in fluorescence was seen (Fig 9B) A bright-field image
of macrophages stimulated with 4b-phorbol 12
myri-state 13 acetate for 12 h is shown in Fig 9C When
4b-phorbol 12 myristate 13 acetate-stimulated cells
were further treated with a Tiron solution (a
cell-per-meable O2Æ scavenger) [20] for 1 h and incubated with
DBZTC for 15 min, fluorescence intensity decreased markedly (Fig 9D) Specificity was confirmed by add-ing a nonenzymatic superoxide scavenger, Tiron The data show that DBZTC is membrane permeable, and able to respond to micromolar changes in O2Æ concen-tration in living cells
Fig 9 Confocal fluorescence and brightfield images of live RAW264.7 macrophages (A) Fluorescence image of RAW264.7 macrophages incubated with DBZTC (10 l M ) at 37 C for 15 min (B) Fluorescence image of probe-stained RAW264.7 macrophages stimulated with 4b-phorbol 12 myristate 13 acetate (2 ngÆmL)1) at 37 C for 12 h (C) Bright-field image of probe-stained RAW264.7 macrophages stimulated with 4b-phorbol 12 myristate 13 acetate (2 ngÆmL)1) at 37 C for 12 h to confirm viability (D) Fluorescence image of cells incubated with
100 l M Tiron at 37 C for 1 h after 4b-phorbol 12 myristate 13 acetate stimulation for 12 h, followed by loading with probe at 37 C for
15 min (scale bar ¼ 15 l M ).
Trang 7In order to further investigate the feasibility of the
proposed method in biological systems, determination
of O2 Æ in cell extracts was performed The fluorescence
intensity of cell extracts treated with a Tiron solution
was used as a blank value The detected O2 Æ mean
content of 4b-phorbol 12 myristate 13
acetate-stimula-ted cell extracts was 0.92· 0.02 lm, based on a
corre-lation between the fluorescence intensity of the extracts
and the regression equation The average recovery test
was carried out using the standard addition method,
and the RSD was obtained from a series of six cell
extracts (Table 1) The results indicated that the
recov-ery and precision of the method applied to determine
O2Æ in the cell extracts were satisfactory
To summarize, the proposed method can be applied
to the quantitative determination of O2 Æ by cell extract
test, and make O2 Æ ‘visible’ in living cells using
confo-cal fluorescence imaging
Conclusion
We have developed a novel fluorescence probe,
DBZTC, that can selectively and dose dependently
detect O2 Æ in cellular systems The probe has good
sta-bility, quick reaction, high sensitivity and exhibits
better selectivity for O2Æ than for other ROS and
biolo-gical compounds (no interference was encountered
from a 500-fold molar excess of H2O2) Furthermore,
DBZTC was confirmed as a cell-permeable probe and
was able to respond to micromolar changes in O2 Æ
con-centration within living cells Overall results establish
the potential value of the probe for facilitating
investi-gations of the generation, metabolism, and mechanisms
of superoxide-mediated cellular homeostasis and injury
Experimental procedures
Apparatus
1
H NMR spectra were recorded on a Bruker Avance 300
Elemental analysis was performed on a Perkin–Elmer Series
II CHNS⁄ O analyzer IR spectra were recorded on PE-983
IR spectrometer (KBr discs cm)1, Perkin–Elmer, Norwalk,
CT) All pH measurements were made using a pH-3c digital
pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass–calomel electrode Fluorimet-ric spectra were obtained with a FLS-920 Edinburgh fluor-escence spectrometer with a xenon lamp and 1.0 cm quartz cells Confocal fluorescence imaging was captured using a Zeiss LSM 510 META scanning microscope containing an Axiopian 2 MOT upright microscope and a 20· water-immersion objective lens Excitation at 488 nm was carried out with an argon ion laser Acquired images were analyzed using image-pro plus 4.5 software
Materials o-Aminobenzenothiol was purchased from Fluka (Shang-hai, China) A stock solution (1 mm) of DBZTC (synthes-ized in-house) was prepared by dissolving in dimethylsulfoxide This stock solution was diluted to 1.00· 10)4m before use The XA solution (1.00 mm) was prepared by dissolving an appropriate amount of XA in 1.00· 10)2m NaOH; XO was from Sigma (St Louis, MO), a stock solution of XO (1.00 UÆmL)1) was prepared
in 2.30 m (NH4)2SO4, 1.00· 10)2m sodium salicylate bio-logy buffer, stored at 2–8C Hepes was from Sigma GSH, superoxide dismutase, tert-butyl hydroperoxide (70% aqueous solution), H2O2 (30% aqueous solution), sodium hypochlorite (NaOCl, 5% aqueous solution), dimethyl-sulfoxide, 3-morpholinosydnonimine hydrochloride, 3-(ami-nopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene, phorbol 12-myristate 13-acetate, and RPMI-1640 medium were from Sigma 1,4-Hydroquinone was from Fluka 4,5-Dihydroxy-1,3-benzenedisulfonic acid disodium salt (Tiron) was from Shanghai Reagent Co Ltd (Shanghai, China) All chemi-cals were of analytical reagent grade, and double-distilled water was used throughout RAW264.7 cells were from American Type Culture Collection (Manassas, VA)
Synthesis and properties of DBZTC 2-chloro-1-formyl-3-hydroxymethylenecyclohexene [21]
A solution of 40.0 mL of dimethylformamide in 40.0 mL of methylene chloride was chilled in an ice bath A solution of 37.0 mL of phosphorus oxychloride in 35.0 mL of methy-lene chloride was added dropwise with stirring Then 10.0 g
of cyclohexanone was added to the mixture The solution was refluxed for 3 h, cooled, poured onto 200 g of ice, and
Table 1 Determination of superoxide anion radicals in cell extracts (n ¼ 6) DBZTC (0.01 m M ), Tiron solution (1 l M ), 4b-phorbol 12 myristate
13 acetate (PMA; 2.0 ngÆL)1), XA (1.00 m M , 30 lL) ⁄ XO (1 U, 30 lL), Hepes (0.10 M , pH 7.40).
Sample
O2–
Æ content of PMA-stimulated cells (l M )
Added (l M )
Found (l M )
Mean (l M )
Average recovery (%)
RSD (%)
1.83, 1.91, 1.96
Trang 8allowed to stand overnight The yellow solid was
crystal-lized from a small volume of acetone cooled with dry ice,
to give 14.50 g (82.39% yield) with a melting point of 130–
131C Anal Calcd for C8H9ClO2: C, 55.67; H, 5.25; Cl,
20.54 Found: C, 55.41; H, 5.43; Cl, 20.47
DBZTC
We added 2.50 g (0.02 mmol) of o-aminobenzenothiol,
1.73 g (0.01 mmol) of
2-chloro-1-formyl-3-hydroxymethyl-enecyclohexene and 50.0 mL of a mixture of 1-butanol and
benzene (7 : 3 v⁄ v) to a 100 mL flask The mixed solution
was heated under reflux for 3 h After the solvent was
removed under reduced pressure, a yellow crude solid was
obtained This was recrystallized from benzene and dried
under a vacuum (2.087 g, 54% yield).1H NMR (300 MHz,
DMSO-d6, 25C, TMS): d 8.10–7.90 (m, 2H, benzol-H),
7.75 (d, 2H, benzol-H), 7.47–7.39 (m, 2H, benzol-H), 7,24
(d, 2H, benzol-H), 4.10 (m, 2H, N-H), 3.12 (m, 2H,
methine-H), 2.3 (m, 3H, cyclohexene-H), 1.15 (m, 4H,
cyclohexene-H) IR (KBr pellet): (cm)1) 3245 (N–H), 3110
(C–H), 1592 (C¼ C) Melting point: 95–97 C Elemental
analysis (%) calcd for C20H19N2S2Cl (found): C 62.10
(62.02), H 4.92 (4.84), N 7.24 (7.36)
Synthesis and characteristics of DBZTC oxide
The oxidization product, DBZTC oxide, was obtained as
follows: 0.050 g DBZTC was dissolved in 10 mL of
eth-anol, 0.20 mL of 2.00· 10)3mKO2solution (0.014 g KO2
was dissolved in 100 mL dimethylsulfoxide) and 20 mL of
buffer solution (pH 7.40) were mixed together, and stirred
for 15 min The solvent was removed, and the residue was
extracted by dichloroethane to give a crude product The
pure brown product was obtained by recrystallization in
ethanol: 1H NMR (90 MHz, DMSO-d6, 25C, TMS): d
8.11–7.93 (m, 2H, benzol-H), 7.66–7.58 (m, 2H, benzol-H),
7.40 (m, 2H, benzol-H), 7,16 (m, 2H, benzol-H), 1.83 (m,
3H, cyclohexene-H), 1.22 (m, 4H, cyclohexene-H) IR (KBr
pellet): (cm)1) 1618 (C¼ N), 1590 (C ¼ C) Elemental
ana-lysis (%) calcd for C20H15N2S2Cl (found): C 62.75 (62.71),
H 3.92 (3.84), N 7.32 (7.37)
Determination of O2–Æ
Into a 10 mL color comparison tube were added 1.00 mL
of DBZTC (1.00· 10)4m), 0.10 mL of XA solution
(1.00 mm), 0.10 mL of XO (1.00 U) and 2.00 mL of Hepes
buffer (0.10 m) in turn After diluted to 10.00 mL volume
with double-distilled water, the mixture was equilibrated
and was laid aside at 37C for 10 min before
determin-ation The fluorescence intensity was measured at kex ⁄ em¼
485⁄ 559 nm against a reagent blank at the same time The
excitation and emission slit were set to 3.5 and 3.5 nm, respectively
Preparation and staining of RAW264.7 macrophages cultures
RAW264.7 macrophages were cultured in Dulbecco’s modi-fied Eagle’s medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at 37C (w/v) in a 5%
CO2⁄ 95% air incubator MCO-15AC (SANYO, Tokyo, Japan) The concentration of counted cells was adjusted to
1· 106
cellsÆmL)1and cells were passed and plated on glass slide at 37C, 5% CO2/95% air for 4 h A set of cells was stimulated with 4b-phorbol 12 myristate 13 acetate (2 ngÆL)1) at 37C for 12 h Some of the stimulated cells were washed with serum-free Dulbecco’s modified Eagle’s medium, and a Tiron solution added (0.1 mm in serum-free Dulbecco’s modified Eagle’s medium, 2.5 mL) After 1 h, cells were washed with Dulbecco’s modified Eagle’s med-ium, and a solution of DBZTC (0.1 mm, 0.2 mL) was added to each dish loaded 2.5 mL serum-free Dulbecco’s modified Eagle’s medium, and incubated for 15 min Before imaging, cover-slips were washed with Hepes (0.10 m,
pH 7.40)
RAW264.7 macrophage extracts RAW264.7 macrophages were cultured in Dulbecco’s modi-fied Eagle’s medium, with the concentration of counted cells at 1· 106
cellsÆmL)1 Some cells were added to a Tiron solution and incubated for 1 h as an O2 scavenger, others were stimulated with 4b-phorbol 12 myristate 13 acetate at 37C for 12 h All cells were then incubated with DBZTC for 30 min at 37C and harvested by centrifuga-tion in the cold, and washed twice with 0.9% NaCl solu-tion These cells were suspended again in a volume of Hepes equal to that in which they had been grown, and dis-rupted for 10 min in a VC 130PB ultrasonic disintegrator (Sonics & Materials Inc., Newtown, CT, USA) During sonic disruption, the temperature was maintained below
4C with circulating ice water The broken cell suspension was centrifuged at 1435 g for 5 min and the pellet discar-ded Cell extracts that had been added to a Tiron solution were divided into six parts The 4b-phorbol 12 myristate 13 acetate-stimulated cell suspension was divided into 12 parts and XA⁄ XO was added into six parts in turn
Acknowledgements
This work was supported by Program for New Cen-tury Excellent Talents in University (NCET-04–0651), the National Natural Science Foundation of China (Nos 20335030 and 20575036)
Trang 91 Chu CT, Levinthal DJ, Kulich SM, Chalovich EM &
DeFranco DB (2004) Oxidative neuronal injury Eur J
Biochem 271, 2060–2066
2 Lowe RD & Snook RD (1991) Sensitive
spectrophoto-metric determination of aluminium using thermal lens
spectrometry Anal Chim Acta 250, 95–104
3 Yi XF & Ben GY (2000) Seasonal variation in
antioxi-dants of Polygonum viviparum and its relation to solar
radiation in alpine meadow Acta Bot Boreal Occident
Sin 20, 201–205
4 Chaudhury S & Sarkar PK (1983) Stimulation of
tubu-lin synthesis by thyroid hormone in the developing rat
brain Biochem Biophys Acta 763, 93–98
5 Fang RZ (1989) Radicals and Enzymes Science Press,
Beijing
6 Kozumbo WJ, Trush MA & Kensler TW (1985) Are
free radicals involved in tumor promotion? Chem Biol
Interact 54, 199–207
7 Nilsson UA, Haraldsson G, Bratell S, Sorensen V,
Akerlund S, Pettersson S, Schersten T & Jonsson O
(1993) ESR-measurement of oxygen radical in vivo after
renal ischemia in the rabbit: effects of pretreatment with
superoxide dismutase and heparin Acta Physiol Scand
147, 263–270
8 Armstead WM, Mirro R, Busija DW & Leffler
CW (1988) Postischemic generation of superoxide
anion by newborn pig brain Am J Physiol 255,
H401–H403
9 Dirnagl U, Lindauer U, Them A, Schreiber S, Pfister
HW, Koedel U, Reszka R, Freyer D & Villringer A
(1995) Global cerebral ischemia in the rat: online
monitoring of oxygen free radical production using
chemiluminescence in vivo J Cereb Blood Flow Metab
15, 929–940
10 Peters O, Back T, Lindauer U, Busch C, Megow D,
Dreier J & Dirnagl U (1998) Increased formation of
reactive oxygen species after permanent and reversible
middle cerebral artery occlusion in the rat J Cereb
Blood Flow Metab 18, 196–205
11 Mu¨nzel T, Afanas’ev IB, Klescchyov AL & Harrison
DG (2002) Detection of superoxide in vascular tissue Arterioscler Thromb Vasc Biol 22, 1761–1768
12 Hatsuo M, Kayko Y, Yoko N, Iho K, Leila H, Narit-sugu U, Shoko Y, Masako F, Yuka F, Yuji Y et al (2005) A design of fluorescent for superoxide based on
a nonredox mechanism J Am Chem Soc 127, 68–69
13 Xu KH, Liu X, Tang B, Yang G, Yang Y & An LG (2006) Design of a phosphinate-based fluorescent probe for superoxide detection in mouse peritoneal
macrophages Eur J Chem 13, 1411–1416
14 Tarpey MM & Fridovich I (2001) Methods of detection
of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite Circ Res 89, 224–236
15 Benov L, Sztejnberg L & Fridovich I (1998) Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical Free Radical Biol Med 25, 826–831
16 Fridovich I (1982) Measuing the activity of superoxide dismutase: an embarrassment of riches In Superoxide Dismutase(Oberley LW, ed.), p 15 CRC Press, Boca Raton, FL
17 Fang YZ & Zheng RL (2002) Theory and Application of Free Radical Biology Scientific Press, Beijing
18 Wilson MR, Stone V, Cullen RT, Searl A, Maynard
RL & Donaldson K (2000) In vitro toxicology of respir-able Montserrat volcanic ash Occup Environ Med 57, 727–733
19 Tang B, Zhang L & Zhang LL (2004) Studies and appli-cation of flow injection spectrofluorimetry based on 2-(2-pyridil)-benzothiazoline as fluorescent probe trapping superoxide anion radicals Anal Biochem 326, 176–182
20 Vaquero EC, Edderkaoui M, Pandol SJ, Gukovsky I & Gukovskaya AS (2004) Reactive oxygen species pro-duced by NAD(P)H oxidase inhibit apoptosis in pan-creatic cancer cells J Biol Chem 279, 34643–34654
21 Reynolds GA & Drexhage KH (1977) Stable hepta-methine pyrylium dyes that absorb in the infrared
J Org Chem 42, 885–888