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Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)
细胞内活性氧的检测(CM-H2DCFDA)   

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Abstract

Reactive oxygen species (ROS) play a critical role in cellular physiopathology. ROS are implicated in cell proliferation, signaling pathways, oxidative defense mechanisms responsible for killing of bacteria, thyroid hormonosynthesis, etc. The cellular Redox homeostasis is balanced by oxidants and antioxidants systems. In several diseases (cancer, neurodegenerative diseases, cardiovascular diseases), the Redox balance is disturbed. In fact, excessive amounts of ROS can damage proteins, lipids and DNA at cellular level.
The choose of a sensitive method for detection of intracellular ROS is very important for detecting the disturbed Redox balance in pathological cells and after exposition of cells to different genotoxic agents (Irradiation, Oxidative stress, etc).
The detection of ROS in biological systems is difficult for several reasons: Method sensibility and probe specificity. The 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) fluorescent probe is commonly employed and may react with several ROS including hydrogen peroxide, hydroxyl radicals and peroxynitrite. The cell-permeant H2DCFDA passively diffuses into cells and is retained in the intracellular level after cleavage by intracellular esterases. Upon oxidation by ROS, the nonfluorescent H2DCFDA is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF). The chloromethyl derivative of H2DCFDA (CM-H2DCFDA) provides much better retention in live cells than H2DCFDA. Dead or dying cells produces ROS. When we want to detect ROS in living cells, we have to stain cells by propidium iodide (PI) and evaluate ROS production only in living cells which are PI negative. In fact, PI intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. PI is excited at 488 nm and emits at a maximum wavelength of 617 nm. Because of these spectral characteristics, PI can be used in combination with other fluorescent probe such as CM-H2DCFDA (excitation/emission: 492–495/517–527 nm).
A probe fluorescence emission can be assessed by Flow cytometry, a standard fluorometer or fluorescence microscopy using appropriate filter.
Flow cytometry is commonly employed to detect intracellular ROS production. Flow cytometry measures fluorescence per cell. The cells is excited by the light source and emitted light from cells are converted to electrical pulses by optical detectors. Emitted Light is send to different detectors by using optical filters: 525 nm Band Pass Filter for FL-1 and 620 nm Band Pass Filter for FL-3. A 525 nm band pass filter (FL-1) placed in the light path prior to the detector will only allow “green” light into the detector. So, FL-1 is used in our protocol to collect green light corresponding to the oxidation of dichlorodihydrofluorescein (DCF) by ROS. FL-1 is the Green (FL-1) channel on flow cytometers. 620 nm Band Pass Filter (FL-3) only allow “red” light into the detector. Red fluorescence emission is measured in the Red (FL-3) channel on most flow cytometers.

Keywords: Reactive oxygen species(活性氧), H2O2(H2O2), Oxidative stress(氧化应激)


Materials and Reagents

  1. 1x Trypsin-EDTA (0.05% / 0.02% in PBS) (PAA Laboratories GmbH, catalog number: L11-004 )
  2. DPBS, Calcium, Magnesium (Life Technologies, InvitrogenTM, catalog number: 14040 )
  3. HBSS Hank’s Balanced Salt Solution (HBSS) with Calcium and Magnesium and without Red Phenol (Life Technologies, InvitrogenTM, catalog number: 14025 )
  4. CM-H2DCFDA (Life Technologies, InvitrogenTM, catalog number: C6827 )
  5. Propidium Iodide - 1.0 mg/ml (Life Technologies, InvitrogenTM, catalog number: P3566 )

Equipment

  1. Cell incubator
  2. Six-well plates
  3. Centrifuge
  4. FACSVantage SE (Becton Dickinson)
  5. 2 x 75 mm, 5 ml polystyrene round bottom test tube (BD Biosciences, catalog number: 352054 )

Procedure

In the protocol described below, we detect the intracellular ROS after exposure of non cancerous human thyroid epithelial cell line “HTori-3.1” to the oxidative stress. HTori-3.1 cells are grown in complete culture medium (see Recipes).

  1. HTori-3.1 cells were plated at a density of 2.5 x 105 cells per well into six-well plates in complete culture medium.
  2. Sixteen hours later, the cells (90% confluence) were washed by warm DPBS, harvested by trypsinization (250 μl of trypsin / Well).
  3. Add 1 ml of complete culture medium (10% FCS) to stop trypsinization and resuspend cells by gently pipetting up and down.
  4. Collect cells into 5 ml polystyrene round bottom tube by centrifugation (5 min at 200 x g) at room temperature and aspirate supernatant.
  5. Wash cell pellet with 4 ml DPBS and centrifuge 5 min at 200 x g at room temperature.
  6. Resuspend cell pellet in HBSS with 10 μmol/L of CM-H2DCFDA and increased concentration of H2O2 (1 – 5 -10 -15 - 20 - 25 and 50 μmol/L) by gently pipetting up and down (500 μl/tube).
  7. Incubate cells (resuspended in tubes) in cell incubator [(37 °C), high relative humidity (95%), and controlled CO2 level (5%)] in the dark for 45 min.
  8. Add Propidium Iodide 0.5 μl/0.5 ml/tube (final concentration = 1 μg/ml) and place the tubes on ice (still in the dark) and proceed immediately to flow cytometry analysis. Note that: a) the propidium iodide staining procedure should be performed immediately before analysis on the flow cytometer, b) when we place tubes on ice we minimize cell activity and we avoid artifactual fluctuations of ROS detection between the first tube analyzed in FACS and following tubes.
  9. A total of 5,000 events are analyzed in flow cytometry.
  10. The cellular viability is assessed by propidium iodide staining. Collect PI fluorescence in the FL-3 channel (620 nm) of FACSVantage SE (Becton Dickinson).
  11. Living cells, which are PI negative, were selected by FACS gating. In these living cells, we record the fluorescence of DCF on FL-1 channel (525 nm) of FACSVantage SE (Becton Dickinson).

Analysis

The effect of H2O2 concentrations from 1 to 50 μmol/L on the intracellular ROS level was determined by flow cytometry analysis. (Figure 1). We can present results as the " all mean fluorescence "or as "M2 percentage" fluorescence variation. M2 indicate the percentage of cells with an increase production of ROS. We measured a concentration-dependent increase of fluorescence, reflecting an increased level of intracellular ROS (Figure 1).



For example, after exposure of cells to 20 μM H2O2, we detect an increase in fluorescence of HTori3.1 cells [Mean fluorescence of all cells is 243.14 in stressed cells and 76.76 in no stressed cells (0 μM H2O2) ]. Our results show that the level of ROS in stressed cells is 3 times more than in no stressed cells.
Also, we can analyze results by "M2 and M1 percentage" fluorescence variation. M1 is placed around the no stressed cells. M2 is placed to the right of M1 to designate positive events. We look at the %Gated for M2: 1.23% (no stressed cells) and 47.12% (cells stressed with 20 μM H2O2). Our results indicate that 47,12% of cells show an increase in ROS level after exposure to 20 μM H2O2.

Recipes

Complete culture medium: phenol red–free RPMI 1640 (Life Technologies, InvitrogenTM), supplemented with 1% (v/v) antibiotics/antimycotics (Life Technologies, InvitrogenTM), 2 mmol/L of L-glutamine (Life Technologies, InvitrogenTM), and 10% (v/v) FCS (PAA Laboratories).

Acknowledgments

This protocol is adapted from Ameziane-El-Hassani et al. (2010).

References

  1. Ameziane-El-Hassani, R., Boufraqech, M., Lagente-Chevallier, O., Weyemi, U., Talbot, M., Metivier, D., Courtin, F., Bidart, J. M., El Mzibri, M., Schlumberger, M. and Dupuy, C. (2010). Role of H2O2 in RET/PTC1 chromosomal rearrangement produced by ionizing radiation in human thyroid cells. Cancer Res 70(10): 4123-4132.

简介

活性氧(ROS)在细胞病理生理学中起关键作用。 ROS涉及细胞增殖,信号通路,负责杀死细菌的氧化防御机制,甲状腺激素合成等。细胞氧化还原稳态由氧化剂和抗氧化剂系统平衡。在几种疾病(癌症,神经变性疾病,心血管疾病)中,氧化还原平衡受到干扰。事实上,过量的ROS可以在细胞水平损伤蛋白质,脂质和DNA。
选择用于检测细胞内ROS的灵敏方法对于检测病理细胞中和在将细胞暴露于不同基因毒性剂(辐射,氧化应激等)之后的干扰的氧化还原平衡是非常重要的。在生物系统中检测ROS是困难的几个原因:方法敏感性和探针特异性。通常使用2',7'-二氯二氢荧光素二乙酸酯(H 2 DCF-DA)荧光探针,并且可与几种ROS反应,包括过氧化氢,羟基和过氧亚硝酸酯。细胞渗透性H 2 DCFDA被动扩散到细胞中,并在细胞内酯酶切割后保留在细胞内水平。在被ROS氧化时,非荧光H 2 DCFDA转化为高度荧光的2',7'-二氯荧光素(DCF)。 H H 2 DCFDA(CM-H 2 DCFDA)的氯甲基衍生物在活细胞中比H H 2 DCFDA提供好得多的保留。死亡或死亡细胞产生ROS。当我们想检测活细胞中的ROS时,我们必须通过碘化丙啶(PI)染色细胞,并仅在PI阴性的活细胞中评估ROS产生。事实上,PI插入到双链核酸中。它被活细胞排除,但可以穿透死亡或死亡细胞的细胞膜。 PI在488nm激发并以617nm的最大波长发射。由于这些光谱特性,PI可以与其它荧光探针例如CM-H 2 2 DCFDA(激发/发射:492-495/517-527nm)组合使用。
A探针荧光发射可以通过流式细胞术,标准荧光计或使用适当过滤器的荧光显微镜来评估。通常使用流式细胞术来检测细胞内ROS产生。流式细胞术测量每个细胞的荧光。细胞被光源激发,并且来自细胞的发射光被光学检测器转换成电脉冲。发射光通过使用光学滤波器发送到不同的检测器:用于FL-1的525nm带通滤波器和用于FL-3的620nm带通滤波器。在检测器之前放置在光路中的525nm带通滤波器(FL-1)将仅允许"绿色"光进入检测器。因此,在我们的协议中使用FL-1来收集对应于通过ROS氧化二氯二氢荧光素(DCF)的绿光。 FL-1是流式细胞仪上的绿色(FL-1)通道。 620 nm带通滤波器(FL-3)只允许"红色"光进入检测器。在大多数流式细胞仪上在红色(FL-3)通道中测量红色荧光发射。

关键字:活性氧, H2O2, 氧化应激


材料和试剂

  1. 1x胰蛋白酶-EDTA(0.05%/0.02%,在PBS中)(PAA Laboratories GmbH,目录号:L11-004)
  2. DPBS,钙,镁(Life Technologies,Invitrogen TM ,目录号:14040)
  3. 具有钙和镁且不含红色酚的HBSS Hank's平衡盐溶液(HBSS)(Life Technologies,Invitrogen TM ,目录号:14025)
  4. CM-H 2 DCFDA(Life Technologies,Invitrogen TM ,目录号:C6827)
  5. 碘化丙啶-1.0mg/ml(Life Technologies,Invitrogen TM,目录号:P3566)

设备

  1. 细胞培养箱
  2. 六孔板
  3. 离心机
  4. FACSVantage SE(Becton Dickinson)
  5. 2×75mm,5ml聚苯乙烯圆底试管(BD Biosciences,目录号:352054)

程序

在下面描述的协议中,我们检测在非癌性人甲状腺上皮细胞系"HTori-3.1"暴露于氧化应激后的细胞内ROS。 HTori-3.1细胞在完全培养基中生长(参见Recipes)。

  1. 将HTori-3.1细胞以2.5×10 5个细胞/孔的密度接种在完全培养基中的六孔板中。
  2. 16小时后,通过温热的DPBS洗涤细胞(90%汇合),通过胰蛋白酶消化收集(250μl胰蛋白酶/孔)。
  3. 加入1毫升完全培养基(10%FCS)停止胰蛋白酶消化和重悬细胞通过轻轻吹吸上下。
  4. 通过在室温下离心(在200×g下5分钟)将细胞收集到5ml聚苯乙烯圆底管中,并抽吸上清液。
  5. 用4ml DPBS洗涤细胞沉淀,并在室温下以200×g离心5分钟。
  6. 将细胞沉淀物在HBSS中用10μmol/L的CM-H 2 DCFDA和增加的H 2 O 2 sub(1 - 5 - 10 -15 - 20 - 25和50μmol/L),通过轻轻吹打上下(500μl/管)。
  7. 将细胞(重悬于管中)在黑暗中孵育45分钟,在细胞培养箱[(37℃),高相对湿度(95%)和控制的CO 2级(5%)] br />
  8. 加入碘化丙啶0.5μl/0.5ml /管(终浓度=1μg/ml)并将管置于冰上(仍然在黑暗中),并立即进行流式细胞术分析。注意:a)碘化丙啶染色程序应当在流式细胞仪上分析之前立即进行,b)当我们将管置于冰上时,我们使细胞活性最小化,并且避免FACS中分析的第一个管之间的ROS检测的伪影波动, 随后管。
  9. 在流式细胞术中分析总共5,000个事件。
  10. 通过碘化丙啶染色评估细胞存活力。在FACSVantage SE(Becton Dickinson)的FL-3通道(620nm)中收集PI荧光。
  11. 通过FACS门控选择PI阴性的活细胞。在这些活细胞中,我们记录DCF在FACSVantage SE(Becton Dickinson)的FL-1通道(525nm)上的荧光。

分析

通过流式细胞术分析测定1至50μmol/L的H 2 O 2 subsubs浓度对细胞内ROS水平的影响。 (图1)。我们可以将结果表示为"所有平均荧光"或"M2百分比"荧光变化。 M2表示具有增加的ROS产生的细胞的百分比。我们测量荧光的浓度依赖性增加,反映细胞内ROS水平增加(图1)。



例如,在将细胞暴露于20μMH 2 O 2 2后,我们检测到HTori3.1细胞的荧光增加[所有细胞的平均荧光在压力下为243.14在无应激细胞(0μMH 2 O 2 O 2)中为76.76。我们的结果表明,应激细胞中ROS的水平是没有应激细胞的3倍 此外,我们可以通过"M2和M1百分比"荧光变化分析结果。 M1放置在无应力单元周围。 M2放置在M1的右边,以指定正事件。我们观察M2的门控百分比:1.23%(无应激细胞)和47.12%(用20μMH 2 O 2 O 2应激的细胞)。我们的结果表明,47.12%的细胞在暴露于20μMH 2 O 2 O 2后显示出ROS水平的增加。

食谱

完全培养基:补充有1%(v/v)抗生素/抗真菌剂(Life Technologies,Invitrogen TM)的无酚红RPMI 1640(Life Technologies,Invitrogen ),2mmol/L的L-谷氨酰胺(Life Technologies,Invitrogen )和10%(v/v)FCS(PAA Laboratories)。

致谢

该协议改编自Ameziane-El-Hassani等人(2010)。

参考文献

  1. Ameziane-El-Hassani,R.,Boufraqech,M.,Lagente-Chevallier,O.,Weyemi,U.,Talbot,M.,Metivier,D.,Courtin,F.,Bidart,JM,El Mzibri, ,Schlumberger,M。和Dupuy,C。(2010)。 H 2 O 2 的作用 RET/PTC1染色体重排由人甲状腺细胞中的电离辐射产生。癌症研究70(10):4123-4132。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ameziane-El-Hassani, R. and Dupuy, C. (2013). Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA). Bio-protocol 3(1): e313. DOI: 10.21769/BioProtoc.313.
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Maria Kauppi
The Walter and Eliza Hall Institute
Hi There

Thank you for your protocol. Do you know if it's possible to stain cells (surface antibody staining on ice for more detailed FACS analysis) after CM-H2DCFDA incubation? Would ROS staining be still reliable after tubes sitting on ice for an extra hour.

Thank you

Maria
9/28/2014 7:47:49 PM Reply
Amy Bazuaye
UTMB
Thanks a lot.
Please can you share with me how you reconstituted your CM-H2DCFDA to a concentration of 10 µmol/L. I have a vial of 50 µg of CM-H2DCFDA
Thank you
6/6/2014 2:33:27 PM Reply
Bio-protocol Editorial Team
bio-protocol.org

Hi Amy,

To solve your problem, you can find out molecular formula (C27H19Cl3O8) molecular weight (577.8013)at http://www.lifetechnologies.com/order/catalog/product/C6827.

Hope it helps.

Bio-protocol Editorial Team

6/9/2014 4:49:51 PM