Simultaneous measurement of reactive oxygen species and reduced glutathione using capillary electrophoresis and laser-induced fluorescence detection in cultured cell lines.

A capillary zone electrophoretic (CZE) method coupled with laser-induced fluorescence (LIF) was developed for the simultaneous determination of two important intracellular parameters related to oxidative stress (i.e. reactive oxygen species, ROS, and reduced glutathione, GSH). This rapid and sensitive method was applied to the study of oxidative stress in cultured V79 fibroblast cells. The fluorogenic reagents selected were: (i) dihydrorhodamine-123 (DHR-123) which is converted intracellularly by ROS to the fluorescent rhodamine-123 dye (Rh-123), and (ii) naphthalene-2,3-dicarboxaldehyde (NDA), which reacts quickly with GSH in cell extracts to produce a fluorescent adduct. Separation of Rh-123, GSH-NDA and gamma-glutamylcysteine-NDA adducts was performed using an uncoated fused-silica capillary and a 100 mM borate buffer, pH 9.2, at 20 degrees C and at an applied voltage of 25 kV; LIF detection was operated using an argon laser. The cell line was also tested for its ability to alleviate oxidative stress induced by tert-butylhydroperoxide (t-BuOOH). Exposure to t-BuOOH (up to 3 mm for 2 h) did not affect the intracellular ROS and GSH concentrations. At higher (4-10 mM) t-BuOOH concentrations, an inverse relationship between the concentrations of ROS and GSH was obtained, showing that the present method can readily evaluate the gradual consumption of the primary cellular scavenger of ROS which occurs simultaneously with the increase of oxidative insult.     

High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel

A novel high-throughput method is presented based on fluorescence images of cells in a microchannel for determination of glutathione (GSH) and reactive oxygen species (ROS) inside single cells. We first present a method to determine GSH and ROS separately, in which GSH in cells is derivatized by 2,3-naphthalenedicarboxaldehyde (NDA), and intracellular ROS is labeled using dihydrorhodamine 123. The cells with either fluorescent derivatized GSH or fluorescent labeled ROS are introduced into a microchannel and fluorescence images of every moving cell in the microchannel are taken continuously using a highly sensitive thermoelectrically cooled electron-multiplying CCD. The fluorescence intensities of the images correspond to the masses of GSH or ROS. An average detection rate of 80-120 cells/min is achieved. We then propose a method for simultaneously determining GSH and ROS, in which ROS is first labeled in the cells. The labeled cells are then introduced into the whole channel and allowed to immobilize onto the glass substrate. The fluorescence images of all the cells in the channel are taken. NDA is then introduced into the channel to derivatize the GSH in the immobilized cells, and fluorescence images of all cells are taken again. An average analysis rate of 20 cells/min is achieved. The masses of GSH and ROS in the single cells can be obtained from the fluorescence intensities of the images using their calibration curves. Since the cells are not lysed, there is no problem with adsorption of biological macromolecules and cellular debris on the channel wall, so that channel treatment, necessary in usual single-cell analysis techniques using CE and microchip electrophoresis, is no longer necessary. For single global cells, this method can also be used to determine the concentrations of ROS and GSH, which has not been reported previously. The concentrations of ROS and GSH in single global cells can be calculated from the determined masses and the cell volume (derived from the diameter of the round fluorescence image of the derivatized GSH). For gastric cancer cells, the concentrations of GSH and ROS are in the range 0.35x10(-3)-1.3x10(-3) mol/L and 0.77x10(-) (6)-1.5x10(-6) mol/L, respectively.     

芯片毛细管电泳评估脂质体介导超氧化物歧化酶减低细胞内氧化应激

超氧化物歧化酶(SOD)可用作抗氧化的药物。它能催化并清除细胞内的活性氧组分(ROS),保护细胞免受自由基的氧化破坏。但是由于SOD分子量较大,难以透过细胞膜进入细胞内,显著降低了SOD的药效。本研究用激光共聚焦荧光显微镜拍摄的荧光图像说明,纳米脂质体可介导SOD进入细胞。用芯片毛细管电泳激光诱导荧光分析法(MCE-LIF)测定单细胞中ROS和谷胱甘肽(GSH)的荧光信号强度,评估了用脂质体包裹的SOD与细胞作用的抗氧化效果。用脂质体包裹的SOD与肝癌细胞共培养2h,与直接用SOD作用于肝癌细胞相比较,细胞内ROS明显降低,GSH明显提高。实验结果说明,用脂质体包裹SOD是一种减低细胞内氧化应激的有效给药途径。     

Determination of reactive oxygen species in single human erythrocytes using microfluidic chip electrophoresis

Reactive oxygen species (ROS) are known to not only mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential if we wish to understand the mechanisms of cellular alterations. In this paper, a microfluidic chip-based approach to the determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip with integrated operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser-induced fluorescence (LIF) detection. Non-fluorescent dihydrorhodamine 123 (DHR 123), which can be oxidized intracellularly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as the fluorogenic reagent. The effect of pH on the migration time of Rh 123 and detection sensitivity was discussed. The present method minimized dilution of intracellular ROS during reaction with DHR 123 and determination. As a result, an extremely low detection limit of 0.8 amol has been achieved. The time required for complete analysis of one human erythrocyte was less than 3 min. A migration time precision of 4.1% RSD was obtained for six consecutively-injected cells. Upon stimulation with 4 mmol/l H₂O₂ for 10 min, the intracellular ROS concentration was found to increase on average by about a factor of 8.4.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.     

Ultrasensitive determination of intracellular superoxide in individual HepG2 cells by microfluidic chip electrophoresis

A microchip electrophoresis method was established for the determination of intracellular superoxide (O₂⁻) in individual HepG2 cells. Dihydroethidium (DHE) was used as the specific fluorescent probe to react with intracellular O₂⁻ to form the fluorescent 2-hydroxyethidium. Excellent resolution between 2-hydroxyethidium and ethidium cation (E⁺) can be achieved within 20 s. E⁺ was reported to be generated from photochemical oxidation of DHE and interfere the determination of O₂⁻ with fluorescence microscopic technique. An extremely low detection limit of 2.0 amol was achieved owing to the minute sample volume and insignificant dispersion effect during microfluidic chip-based electrophoretic separation. Furthermore, only 2-hydroxyethidium peak was detected with the suggested single-cell analysis method, which indicates the photooxidation of DHE to E⁺ could be blocked by isolating either oxygen or light from them.     

Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells

A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells.     

Construction of a Selective Fluorescent Probe for GSH Based on a Chloro‐Functionalized Coumarin‐enone Dye Platform

Glutathione (GSH), the most abundant intracellular biothiol, protects cellular components from damage caused by free radicals and reactive oxygen species (ROS), and plays a crucial role in human pathologies. A fluorescent probe that can selectively sense intracellular GSH would be very valuable for understanding of its biological functions and mechanisms of diseases. In this work, a 3,4‐dimethoxythiophenol‐substituted coumarin‐enone was exploited as a reaction‐type fluorescent probe for GSH based on a chloro‐functionalized coumarin‐enone platform. In the probe, the 3,4‐dimethoxythiophenol group functions not only as a fluorescence quencher through photoinduced electron transfer (PET) to ensure a low background fluorescence, but also as a reactive site for biothiols. The probe displays a dramatic fluorescence turn‐on response toward GSH with the long‐wavelength emission (600 nm) and significant Stokes shift (100 nm). The selectivity of the probe toward GSH over cysteine (Cys), homocysteine (Hcy), and other amino acids was demonstrated. Assisted by laser‐scanning confocal microscopy, we have demonstrated that the probe could specifically sense GSH over Cys/Hcy in human renal cell carcinoma SiHa cells.     

A ratiometric fluorescent probe for real-time monitoring of intracellular glutathione fluctuations in response to cisplatin

Real-time imaging of fluctuations in intracellular glutathione (GSH) concentrations is critical to understanding the mechanism of GSH-related cisplatin-resistance. Here, we describe a ratiometric fluorescence probe based on a reversible Michael addition reaction of GSH with the vinyl-functionalized boron-dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) 1. The probe was applied for real-time monitoring of the fluctuations in GSH levels in cells under cisplatin treatment. Notably, in cellular cisplatin-sensitive A549 cells, GSH concentrations rose until cell death, while in cisplatin-resistant cell lines, GSH levels first rose to the maximum then fell back to the initial concentration without significant apoptosis. These results indicate that different trends in GSH fluctuation can help distinguish cisplatin-resistant from cisplatin-sensitive cells. As such, this study has shown that probe 1 may potentially be used for real-time monitoring of intracellular GSH levels in response to therapeutics.

Real-time imaging of intracellular glutathione in response to cisplatin by a ratiometric fluorescent probe reveals that the different trends in intracellular GSH levels is crucial in distinguishing cisplatin-resistant from cisplatin-sensitive cells.     

Glutathione modulates the level of free radicals produced in UVA-irradiated cells

We have developed an assay to detect reactive oxygen species (ROS) generated by UVA radiation utilising chemical probes which become fluorescent upon oxidation. Using a human bladder carcinoma cell line (MGH-U1) and spontaneously immortalised keratinocytes (HaCaT), we have shown a UVA (narrow band 365+/-5 nm) dose-dependent increase in fluorescence by flow cytometry following loading of the cells with either dihydrorhodamine 123 (DHR) or 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The UVA response of both DHR and DCFH was enhanced by elevation of intracellular levels of the photosensitiser protoporphyrin IX by incubation for 2.5 h with 5-aminolaevulinic acid. Depletion of the antioxidant glutathione (GSH) using the inhibitor D,L-buthionine-sulphoximine (BSO), resulted in an increase in the UVA-induced fluorescence of DCF but not of rhodamine 123. Conversely, raising intracellular GSH levels with N-acetyl cysteine (NAC) had relatively little protective effect in terms of degree of induced fluorescence.     

Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin

Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280-320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m(-2) UVB), the model finds that 14.70 x 10(-3) mol of ROS that react with DHR are generated in the stratum corneum of an average adult-size face (258 cm(-2)). Approximately 10(-4) mol are potentially generated in the lower epidermal strata. The data show that two-photon fluorescence imaging can be used to detect ROS in UV-irradiated skin.     

A nuclear-localized fluorescent hydrogen peroxide probe for monitoring sirtuin-mediated oxidative stress responses in vivo

Hydrogen peroxide (H(2)O(2)) can serve as a beneficial signaling agent or toxin depending on its concentration and location within a cell or organism. Methods to measure the localized accumulation of H(2)O(2) in living specimens remain limited. Motivated to meet this need, we have developed a nuclear-localized fluorescent probe for H(2)O(2), Nuclear Peroxy Emerald 1 (NucPE1), to selectively interrogate ROS fluxes within this sensitive organelle. NucPE1 selectively accumulates in the nuclei of a variety of mammalian cell lines as well as in whole model organisms like Caenorhabditis elegans, where it can respond to subcellular changes in H(2)O(2) fluxes. Moreover, in?vivo NucPE1 imaging reveals a reduction in nuclear H(2)O(2) levels in worms overexpressing sir-2.1 compared with wild-type congeners, supporting a link between this longevity-promoting sirtuin protein and enhanced regulation of nuclear ROS pools.     

Scanning electrochemical microscopy studies of glutathione-modified surfaces. An erasable and sensitive-to-reactive oxygen species surface

A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the -SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O(2) to superoxide, O(2)(?-), near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O(2)(?-) and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O(2)(?-) at the tip probe electrode.     

MULTIPLE MODALITY TREATMENT OF CARCINOMA CELLS WITH Pt(Rh-123)2 PLUS X-RAY PLUS LIGHT

A complex of platinum tetrachloride with two molecules of rhodamine-123 (Rh-123), Pt(Rh-123)2, has been reported to act as hypoxic cell radiosensitizer of carcinoma cells in vitro and in vivo. In the present paper we report that Pt(Rh-123)2 photosensitizes human mammary carcinoma (MCF-7) cells and cis-platinum resistant human mammary carcinoma (MCF-7/CP) cells to 400-800 nm light in vitro. The efficiency of photosensitization by Pt(Rh-123)2 was 10 times greater than for Rh-123. Combination therapy using Pt(Rh-123)2 plus x-ray plus light was also much more effective compared to the combination therapy using Rh-123 plus x-ray plus light. After 15 microM of Rh-123 plus x-ray (0-8 Gy) plus light (5 J/cm2) treatment, cell survival curve was parallel to the x-ray cell survival curve with an initial decrease in the surviving fraction corresponding to the drug plus light mediated killing. Cell killing caused by Rh-123 (15 microM) plus x-ray (0-8 Gy) plus light (5 J/cm2) was additive as determined by the product of the surviving fraction after Rh-123 plus light and x-ray. In contrast, for 15 microM of Pt(Rh-123)2 plus x-ray (8 Gy) plus light (5 J/cm2) treatment, whereas additive killing predicts a survival fraction of approximately 0.024, in reality, the combination therapy caused the survival fraction to decrease to 0.0012, implying that the cell killing was enhanced by a factor of 20. Using Pt(Rh-123)2 plus x-ray plus light, supra-additive cell killing was also observed under hypoxic conditions, although compared to normally oxygenated conditions the degree of cytotoxicity was significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrochemical antioxidant detection technique based on guanine-bonded graphene and magnetic nanoparticles composite materials

An antioxidant (AO) amperometric technique based on guanine attached to graphene and Fe(3)O(4) nanoparticles (NPs) magnetic materials was developed. Guanine molecules acted as an antioxidant competitor were bonded with graphene nanosheets, onto which magnetic Fe(3)O(4) NPs were attached and the as-prepared magnetic composite can be attracted to the electrode surface by an external magnetic field. When applied with negative potentials, the dissolved oxygen was reduced to H(2)O(2) at the electrode surface, and then reacted with the EDTA-Fe(ii) complex via a Fenton-like reaction to produce OH radicals. After oxidation damage by OH radicals, the electrochemical oxidation of guanine gave a decreased current. In the presence of AOs, the reactive oxygen species (ROS, e.g. OH radicals and H(2)O(2)) were scavenged by AOs and fewer guanine probe molecules were oxidized, thus inducing a higher electrochemical oxidation current of guanine. So AOs competed with the guanine probe molecules toward oxidation by ROS. The current signals of the guanine probe molecules were proportional to the concentrations of AOs. A kinetic model was proposed to quantify the ROS scavenging capacities of the AOs. Using guanine as an oxidizable probe and OH radicals and H(2)O(2) as endogenous ROS, this kind of AO detection technique mimicks the antioxidant protection mechanism by small AO molecules in the human body.     

Indirect detection of photosensitizer ex vivo

Photodynamic therapy induces the production of reactive oxygen species (ROS) within tissues exposed to laser light after administration of a sensitizer. In the context of continuing clinical and commercial development of chemicals with sensitizing properties, a minimally invasive assay is needed to determine the tissue kinetics of fluorescent or non-fluorescent photoreactive drugs. The level of ROS was determined ex vivo from 1 mm3 biopsy samples using 2'-7' dichlorofluorescin diacetate (DCFH-DA), a fluorescent probe which was converted into highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This assay was tested on meta(tetrahydroxyphenyl)chlorin (m-THPC, FOSCAN), a powerful and fluorescent sensitizer, and bacteriochlorophyll derivative WST09 (TOOKAD), a near-infrared absorbing sensitizer that is only slightly fluorescent. In conjunction with the ROS assay, the tissue accumulation of m-THPC was determined on biopsy samples using an optic fibre spectrofluorometer (OFS). DCF fluorescence was proportional to the level of oxidation induced by horseradish peroxidase used as a control and to the concentration (range: 0-5 microg x ml(-1)) of both selected photosensitizers irradiated in a tube together with DCFH. Regardless of the organ studied, an excellent correlation was found between fluorescence measurement by OFS and ROS determination for m-THPC. m-THPC (2 mg x kg(-1) iv) accumulation in tumour tissues was best after 48 h, and the best signal was obtained in liver. With non-fluorescent WST09 (2 mg x kg(-1)), ROS determination showed the best tumour uptake 48 h after injection, with a tumour/muscle ratio of 5.4. The ROS assay appears to be feasible for determining sensitizer concentration in regular grip biopsy tissue samples.     

Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes

We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (UVA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H2O2), detected by a fluorescent probe carboxydichlorodihydrofluorescein, and caused oxidative DNA damage, single strand-breaks and alkali-labile sites, measured by alkaline single cell gel electrophoresis (comet assay). Superoxide anion (O2*-) was a likely substrate for H2O2 production since diethyldithiocarbamate (DDC), a superoxide dismutase blocker, decreased the level of intracellular H2O2. Hydrogen peroxide was shown to play a central role in DNA damage. Increasing the intracellular levels of H2O2 with aminotriazole (AT) (a catalase blocker) and buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis) potentiated the UVA-induced DNA damage. Exogenous H2O2 was also able to induce DNA damage. Since H2O2 alone is not able to damage DNA directly, we investigated the significance of the H2O2-derived hydroxyl radical (*OH). Addition of FeSO4, that stimulates *OH formation from H2O2 (Fenton reaction) resulted in a twofold increase of DNA-damage. Desferrioxamine, an iron chelator that blocks the Fenton reaction, prevented UVA-induced DNA damage. We also employed a panel of less specific antioxidants and enzyme modulators. Sodium selenite (Na-Se) present in glutathione peroxidase and thioredoxin reductase and addition of glutathione (GSH) prevented DNA-damage. Tocopherol potently prevented UVA-and H2O2-induced DNA damage and reduced intracellular H2O2 -levels. Ascorbic acid reduced H2O2 production, but only partly prevented DNA damage. Singlet oxygen (1O2) did not seem to play an important role in the UVA-induced DNA-damage since the specific 1O2 scavenger sodium azide (NaN3) and the less specific 1O2 scavenger beta-carotene did not markedly prevent either DNA-damage or H2O2 production. In conclusion the conversion of H2O2 to *OH appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H2O2 appears to originate from O2*- generated by UVA irradiation.     

SERS纳米探针对电刺激过程中细胞内产生活性氧的检测

电刺激是用于细胞内紊乱电活动引起疾病的一类重要治疗方式. 在电刺激过程中是否会诱导细胞内活性氧(ROS)水平的改变, 以及常规抗氧化抑制药物与电刺激治疗同时运用带来的影响, 目前尚未有相关研究. 本文设计了一种具有较好生物相容性的金/银核壳纳米棒表面增强拉曼(SERS)活性探针, 用于电刺激过程中细胞内产生ROS的检测. 将该探针与细胞共孵育, 使其内化入细胞, 对细胞进行不同时间的电刺激, 利用拉曼光谱对SERS探针的信号进行检测. 实验结果表明, 随着电刺激时间的延长, SERS信号减弱, 说明细胞内产生ROS的量明显增加. 该传感机制是利用ROS能刻蚀金/银核壳纳米棒的银壳, 从而使其变薄引起SERS信号减弱. 抗坏血酸(AA)和谷胱甘肽(GSH)两种抗氧化抑制剂类药物与电刺激同时运用时, 可观察到它们会对电刺激过程产生的ROS有清除作用. 该研究发展了一类用于细胞内ROS检测的光谱方法, 也为异常的氧化应激和肿瘤治疗过程中的组合用药提供了建议.     

Analysis of intracellular reactive oxygen species by micellar electrokinetic capillary chromatography with laser-induced-fluorescence detector

Reactive Oxygen Species (ROS) are an important part of the normal cell growth cycle and play essential roles in many biological functions. Many techniques have been developed to detect and measure the amount of ROS present in cells. These techniques include spectrophotometry, high performance liquid chromatography (HPLC), capillary electrophoresis with laser-induced-fluorescence detector (CE-LIF) and capillary electrophoresis-on-a-chip with LIF. As ROS has a short half-life outside of the cell, various fluorescent probes, such as dihydrorhodamine 123 (DHR 123), that are membrane permeable have been used in the detection of intracellular ROS. In this paper, micellar electrokinetic capillary chromatography (MEKC) was coupled with LIF detector. Fluorescent probe, 3′-(p-aminophenyl)-fluorescein (APF), was used to detect specific ROS in Chinese Hamster Ovary (CHO) suspension cells as well as attached mouse bone marrow-derived dendritic cells (BMDCs). Separation buffer composition was optimized at a concentration of 25?mM tetraborate buffer with 50?mM sodium dodecyl sulfate at pH 9.3 and lysis of the cells was done successfully with a buffer of 70% ethanol and 0.1mM sodium dodecyl sulfate using a cell amount of 1?×?10⁷. ROS in cells was successfully analyzed by MEKC-LIF method developed, with acceptable RSD for time at 1.54% and area at 2.81%.     

Chromanols from Sargassum siliquastrum and their antioxidant activity in HT 1080 cells

Six meroterpenoids (compounds 1-6) of chromene class, including three known compounds (1-3), were isolated from Sargassum siliquastrum. The structure of these compounds was established by extensive 2D-NMR experiments such as (1)H gradient double quantum filtered correlation spectroscopy (gDQCOSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), gradient heteronuclear multiple quantum coherence (gHMQC), and gradient heteronuclear multiple bond correlation (gHMBC), and by comparison with published spectral data. The antioxidant activity of these compounds was evaluated by various antioxidant tests, such as scavenging effects on generation of intracellular reactive oxygen species (ROS), increments of intracellular glutathione (GSH) level, and inhibitory effects on lipid peroxidation in human fibrosarcoma HT 1080 cells. Compounds (1-6) significantly decreased generation of intracellular ROS and inhibited lipid peroxidation while they increased levels of intracellular GSH at a concentration of 5 μg/ml.