Flow Cytometric Detection of Reactive Oxygen Species

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Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal catalyzed oxidation at a controlled rate. However, ROS can be induced under some stress conditions especially exposure to environmental oxidants and certain drugs that leads to oxidative stress. Exceed ROS can cause damages in the building blocks of cells including DNA, proteins, and lipids, and eventually results in cell death. Cell-permeant 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is a widely used ROS indicator. The reduced non-fluorescent fluorescein H2DCFDA can be oxidized and converted into fluorescent 2’, 7’-dichlorofluorescein (DCF) by intracellular ROS. In this protocol, we applied H2DCFDA to label the intracellular ROS and detected the DCF intensity by flow cytometry.

Materials and Reagents

  1. Cells to analyze (this protocol has been successfully performed on A549, CL1-0, and IMR-90 cells)
  2. Dulbecco's Phosphate-buffered saline (DPBS)
  3. 2’,7’-dichlorofluorescein diacetate (H2DCFDA) (Life Technologies, InvitrogenTM, catalog number: D-399 )
  4. Anhydrous DMF (N,N-dimethylformamide) (Sigma-Aldrich, catalog number: 227056 )
  5. N-acetyl-L-cysteine (NAC) (Sigma-Aldrich, catalog number: A7250 )
  6. Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: 349887 )
  7. 5 ml polystyrene BD falcon round-bottom tube with cell strainer cap (BD Biosciences, catalog number: 352235 )
  8. Sodium Chloride (NaCl)                     
  9. Potassium Chloride (KCl)                                       
  10. Potassium Phosphate, monobasic (KH2PO4)                                   
  11. Sodium Phosphate, dibasic (Na2HPO4)
  12. DPBS (see Recipes)
  13. H2DCFDA stock (10 mM) (see Recipes)
  14. NAC stock (1 M) (see Recipes)
  15. H2O2 stock (1 M) (see Recipes)


  1. Flow cytometry
  2. Water bath
  3. Centrifuge


  1. Cells were cultured with complete medium in a 6 cm dish at 37 °C and 5% CO2.
  2. Cells were treated with NAC or H2O2 after reaching 70-90% confluency. ROS scavenger NAC is a precursor to cysteine and glutathione which are strong antioxidants; while H2O2 is a compound with an oxygen-oxygen single bond and is known as a strong oxidizer.
  3. (For negative control) Incubate cells with freshly prepared 5 mM NAC in culture medium for 1 h at 37 °C after three time washes of pre-warmed DPBS.
  4. (For positive control) Incubate cells with freshly prepared 0.1 mM H2O2 from 1 M stock in DPBS for 20 min at 37 °C after three time washes of pre-warmed DPBS.
  5. Dilute the H2DCFDA stock solutions into DPBS to make 0.1 μM working solution.
  6. Cells were harvested by trypsinization.
  7. Suspend cells in working solution at a density of 1 x 106 cells/ml and incubate at 37 °C for 30 min and protect from light.
  8. Centrifuge the tubes at 130 x g for 5 min.
  9. Remove the supernatant and gently resuspend the cells in pre-warmed DPBS.
  10. Repeat the wash steps 8 and 9 twice.
  11. Submit samples to flow cytometry for ROS detection using the 488 nm laser for excitation and detected at 535 nm.


  1. Gate on the main cell population.

    Figure 1. Cells were analyzed according to their size and granularity. The X-axis represents the forward scatter (FSC) parameter which is relative to the size for the cell. The Y-axis shows the side scatter (SSC) parameter which correlates with the components inside the cell. Gate 1 indicates the main population of the cells we analyzed.
  1. Show the intensity of H2DCFDA of cells in gate 1.

    Figure 2. Histogram of H2DCFDA. It shows how many cells are at each intensity of H2DCFDA. The X-axis represents the H2DCFDA intensity, while the Y-axis indicates the cell counts in corresponding fluorescence intensity.


  1. DPBS (1 L)
    8 g Sodium Chloride (NaCl)
    0.2 g Potassium Chloride (KCl)
    0.2 g Potassium Phosphate, monobasic (KH2PO4)
    1.15 g Sodium Phosphate, dibasic (Na2HPO4)
    Adjust to pH = 7.3.
  2. H2DCFDA stock (10 mM)
    Dissolve 10 mg in 2.05 ml DMF to make 10 mM stock.
    Aliquot and store at -20 °C.
    Avoid from light and repeated freeze/thaw cycles.
  3. NAC stock (1 M)
    Dissolve 1 g in 6.128 ml distilled water to make 1 M stock.
    Filter, aliquot and store at -20 °C.
    Avoid from light and repeated freeze/thaw cycles.
  4. H2O2 stock (1 M)
    Dilute 50 μl H2O2 from 35% (=11.6M) in 530 μl sterile deionized water to make 1 M stock.


  1. Chang, H. Y., Huang, H. C., Huang, T. C., Yang, P. C., Wang, Y. C. and Juan, H. F. (2012). Ectopic ATP synthase blockade suppresses lung adenocarcinoma growth by activating the unfolded protein response. Cancer Res 72(18): 4696-4706.


活性氧(ROS)是含有羟基自由基的分子或具有不成对电子的过氧化物。 在健康好氧细胞中,ROS作为氧化磷酸化,氧化还原酶或金属催化氧化的副产物以受控速率天然产生。 然而,ROS可以在一些应激条件下诱导,特别是暴露于环境氧化剂和导致氧化应激的某些药物。 Exceed ROS可导致细胞构建块(包括DNA,蛋白质和脂质)的损伤,并最终导致细胞死亡。 细胞渗透剂2',7'-二氯二氢荧光素二乙酸酯(H 2 DCFDA)是广泛使用的ROS指示剂。 还原的非荧光荧光素H 2 DCFDA可以被氧化并通过细胞内ROS转化为荧光2',7'-二氯荧光素(DCF)。 在该协议中,我们应用H 2 DCFDA标记细胞内ROS并通过流式细胞术检测DCF强度。


  1. 要分析的细胞(此协议已在A549,CL1-0和IMR-90细胞上成功进行)
  2. Dulbecco磷酸盐缓冲盐水(DPBS)
  3. 2',7'-二氯荧光素二乙酸酯(H 2 DCFDA)(Life Technologies,Invitrogen TM,目录号:D-399)
  4. 无水DMF(N,N-二甲基甲酰胺)(Sigma-Aldrich,目录号:227056)
  5. N-乙酰基-L-半胱氨酸(NAC)(Sigma-Aldrich,目录号:A7250)
  6. 过氧化氢(H 2 O 2)(Sigma-Aldrich,目录号:349887)
  7. 5ml具有细胞过滤帽的聚苯乙烯BD falcon圆底管(BD Biosciences,目录号:352235)
  8. 氯化钠(NaCl)                  
  9. 氯化钾(KCl)                    ;                 
  10. 磷酸二氢钾(KH 2 PO 4)                                     
  11. 磷酸二钠(Na 2 HPO 4)
  12. DPBS(参见配方)
  13. H 2 DCFDA原液(10mM)(参见配方)
  14. NAC库存(1 M)(参见配方)
  15. (参见配方)


  1. 流式细胞术
  2. 水浴
  3. 离心机


  1. 在37℃和5%CO 2的6cm培养皿中用完全培养基培养细胞。
  2. 在达到70-90%融合后用NAC或H 2 O 2 O 2处理细胞。 ROS清除剂NAC是作为强抗氧化剂的半胱氨酸和谷胱甘肽的前体;而H 2 O 2 O 2是具有氧 - 氧单键的化合物,并且被称为强氧化剂。
  3. (对于阴性对照)在预热的DPBS洗涤三次后,在37℃下将细胞与新鲜制备的5mM NAC在培养基中孵育1小时。
  4. (对于阳性对照)在37℃下,在37℃下,用来自1MBS储备液的新鲜制备的0.1mM H 2 O 2 Sub储存细胞与37℃下孵育细胞20分钟,暖DPBS。
  5. 将H 2 DCFDA储备溶液稀释到DPBS中以制备0.1μM工作溶液。
  6. 通过胰蛋白酶消化收获细胞
  7. 将细胞以1×10 6个细胞/ml的密度悬浮在工作溶液中,并在37℃下孵育30分钟并避光。
  8. 在130×g离心管中5分钟。
  9. 取出上清液,轻轻地将细胞重悬在预热的DPBS中
  10. 重复洗涤步骤8和9两次
  11. 将样品提交流式细胞术,使用488 nm激光进行ROS检测,并在535 nm处检测。


  1. 在主细胞群体上门。

    图1.根据细胞的大小和粒度分析细胞。 X轴表示相对于单元格大小的前向散射(FSC)参数。 Y轴示出与单元内部的分量相关的侧向散射(SSC)参数。门1表示我们分析的细胞的主要群体。
  1. 显示门1中细胞的H 2 DCFDA的强度。

    图2. H的直方图 2 DCFDA。它显示每个强度H 2 DCFDA。 X轴表示H 2 DCFDA强度,而Y轴表示相应荧光强度中的细胞计数。


  1. DPBS(1 L)
    0.2g磷酸二氢钾(KH 2 PO 4)
    1.15g磷酸二氢钠(Na 2 HPO 4)
    调节至pH = 7.3
  2. H 2 DCFDA原液(10mM) 将10mg溶解在2.05ml DMF中以制备10mM储备液。
    等分并储存在-20°C 避免光照和反复冻融。
  3. NAC库存(1 M)
    过滤,分装并储存在-20°C 避免光照和反复冻融。

  4. 。 在530μl无菌去离子水中从35%(= 11.6M)稀释50μlH 2 O 2 2以制备1μM母液。


  1. Chang,H.Y.,Huang,H.C.,Huang,T.C.,Yang,P.C.,Wang,Y.C.and Juan,H.F.(2012)。 异位ATP合酶阻断通过激活解折叠蛋白反应来抑制肺腺癌生长 Cancer Res 72(18):4696-4706。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chang, H., Huang, H., Huang, T., Yang, P., Wang, Y. and Juan, H. (2013). Flow Cytometric Detection of Reactive Oxygen Species. Bio-protocol 3(8): e431. DOI: 10.21769/BioProtoc.431.

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Rodrigo Hoyos
Boston Children's Hospital
Hi, I'm trying to set up your protocol and I wanted to confirm if NAC/H2O2 were present in the cell culture media while the cells were getting to a confluency of 70-90% (for positive and negative controls respectively) and after cells were treated for an adittional hour. (Steps 2, 3 and 4 of your protocol). Thanks for your help.
12/3/2015 8:29:54 AM Reply
Hsin-Yi Chang
Kyoto University

Hi, I'm not sure what your question is. According to the protocol, cells were treated with NAM or H2O2 after reaching 79-90% confluency. At the end of treatment, cells were trypsinized and incubated with dye containing DPBS.

12/4/2015 3:32:51 AM

Rodrigo Hoyos
Boston Children's Hospital

Thanks a lot for your answer, that's what i figured but I got confused since in step two it says "Cells were treated with NAC or H2O2 until 70-90% confluency reached" instead of after reaching 70-90% confluency.

12/4/2015 5:40:30 AM