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Flow Cytometric Detection of Mitochondrial Membrane Potential
流式细胞术检测线粒体膜电位   

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Abstract

Mitochondrial membrane potential (Δψm) is an important parameter of mitochondrial function and an indicator of cell health. Depletion of Δψm suggests the loss of mitochondrial membrane integrity reflecting the initiation of the proapoptotic signal. Recently, lipophilic cationic fluorescent dyes have been developed to detect Δψm by accumulating in the mitochondrial matrix until the Nernstian equilibrium distribution of lipophilic cations is reached. In this protocol, we applied a cell-permeant, green-fluorescent, lipophilic dye 3,3'-dihexyloxacarbocyanine Iodide (DiOC6(3)) which accumulates in mitochondria due to their large negative membrane potential, it can be applied to monitor the mitochondrial membrane potential using flow cytometric detection.

Materials and Reagents

  1. Cells to analyze (this protocol has been successfully performed on A549, CL1-0, IMR-90, and MCF7 cells)
  2. Dulbecco's Phosphate-Buffered Saline (DPBS)
  3. DiOC6(3) (Life Technologies, Molecular Probes®, catalog number: D-273 )
  4. DMSO (Sigma-Aldrich, catalog number: D8418 )
  5. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  6. 5 ml polystyrene BD falcon round-bottom tube with cell strainer cap (BD Biosciences, catalog number: 352235
  7. Sodium Chloride (NaCl)
  8. NaOH
  9. Potassium Chloride (KCl)    
  10. Potassium Phosphate, monobasic (KH2PO4)  
  11. Sodium Phosphate, dibasic (Na2HPO4)
  12. DPBS (see Recipes)
  13. DiOC6 stock (see Recipes)
  14. Paraformaldehyde stock (8%) (see Recipes)

Equipment

  1. Flow cytometry
  2. Water bath with temperature control
  3. Centrifuge
  4. 6-cm dish
  5. 0.22 μm filter

Procedure

  1. Cells were cultured with complete medium in a 6 cm dish at 37 °C and 5% CO2.
  2. Cells were harvested until 70-90% confluency reached.
  3. (For positive control of depletion of mitochondria membrane potential) Suspend 1 x 106 cells in 1 ml freshly prepared 4% paraformaldehyde diluted from stock using DPBS for 10 min at room temperature and wash them with 1 ml pre-warmed (37 °C) DPBS three times.
  4. Dilute the DiOC6 stock solutions into DPBS to make 0.1 μM working solution.
  5. Suspend cells at a density of 1 x 106 cells/ml in dye working solution and protect from light.
  6. Incubate the cells at 37 °C for 15 min.
  7. Centrifuge the tubes at 130 x g for 5 min.
  8. Remove the supernatant and gently resuspend the cells in 1 ml pre-warmed DPBS.
  9. Repeat the wash steps 7 and 8 twice.
  10. Submit samples to flow cytometry for mitochondrial membrane potential measurement.

Data analysis

  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 DiOC6(3) of cells in gate 1.


    Figure 2. Histogram of DiOC6(3). It shows how many cells are at each intensity of DiOC6(3). The X-axis represents the DiOC6(3) intensity, while the Y-axis indicates the cell counts in corresponding fluorescence intensity.

  1. Overlap the DiOC6(3) signals of positive control which indicating the population of depletion of mitochondria membrane potential (MMP).


    Figure 3. Overlapped histograms of healthy cells (red) and cells loss of mitochondria membrane potential (gray). DiOC6(3) histograms of healthy cells and cells loss of MMP were overlapped to compare the intensity differences.

  1. Gate on cells loss of MMP (R1) and healthy cells (R2).


    Figure 4. Selection of cell populations in health (R2) or depletion of MMP (R1). Healthy cells with high intensity in DiOC6(3) were gated in R1, while cells loss of MMP with lower DiOC6(3) intensity were gated in R1.

Recipes

  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. DiOC6 stock (10 mM)
    Dissolve 10 mg in 1.747 ml DMSO to make 1 mM stock.
    Aliquot and store at -20 °C.
    Avoid from light and repeated freeze/thaw cycles.
  3. Paraformaldehyde stock (8%)
    Weight 8 g paraformaldehyde to 90 ml distilled water (in a fume hood).
    Add 0.1 ml of 10 N NaOH.
    Heat and stir the solution until the granules are fully dissolved (do not heat the solution above 65 °C).
    Turn off the heater and adjust to pH 7.4 with about 0.3 ml of 20% HCl.
    Bring volume to 100 ml with distilled water.
    Sterilize the solution with 0.22 μm filter and can be stored at 4 °C for 30-60 days.

References

  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.

简介

线粒体膜电位(Δψm)是线粒体功能的重要参数和细胞健康的指标。 Δψm的消耗暗示线粒体膜完整性的丧失反映了促细胞凋亡信号的启动。 最近,已经开发了亲脂性阳离子荧光染料来通过在线粒体基质中积累来检测Δψm,直到达到亲脂阳离子的能斯特平衡分布。 在这个协议中,我们应用细胞渗透,绿色荧光,亲脂染料3,3'-二己氧基碳菁碘化物(DiOC6(3))积累在线粒体,由于他们大的负膜电位,它可以应用于监测线粒体 膜电位使用流式细胞术检测。

材料和试剂

  1. 要分析的细胞(此协议已在A549,CL1-0,IMR-90和MCF7细胞上成功实施)
  2. 达尔伯克磷酸盐缓冲盐水(DPBS)
  3. DiOC 6(3)(Life Technologies,Molecular Probes ,目录号:D-273)
  4. DMSO(Sigma-Aldrich,目录号:D8418)
  5. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  6. 5ml具有细胞过滤帽的聚苯乙烯BD falcon圆底管(BD Biosciences,目录号:352235)
  7. 氯化钠(NaCl)
  8. NaOH
  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. DiOC6股票(见配方)
  14. 多聚甲醛原料(8%)(参见配方)

设备

  1. 流式细胞术
  2. 带温度控制的水浴
  3. 离心机
  4. 6厘米的菜
  5. 0.22μm过滤器

程序

  1. 在37℃和5%CO 2的6cm培养皿中用完全培养基培养细胞。
  2. 收获细胞直至达到70-90%融合
  3. (用于线粒体膜电位消耗的阳性对照)在室温下使用DPBS在1ml新鲜制备的4%多聚甲醛(从原液中稀释10分钟)中悬浮1×10 6个细胞,并用1ml前 (37℃)DPBS三次。
  4. 将DiOC 6储备溶液稀释到DPBS中以制备0.1μM工作溶液。
  5. 在染料工作溶液中以1×10 6个细胞/ml的密度悬浮细胞,并避免光照。
  6. 在37℃孵育细胞15分钟。
  7. 在130×g离心管子5分钟。
  8. 取出上清液,轻轻地将细胞重悬在1ml预热的DPBS中
  9. 重复洗涤步骤7和8两次
  10. 将样品提交至流式细胞术进行线粒体膜电位测量

分析

  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轴表示相对于细胞大小的前向散射(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中单元格的DiOC <6>(3)的强度。


    图2. DiOC直方图 6 (3)。 它显示在每种强度的DiOC6(3)下有多少个细胞。 X轴表示DiOC6(3)强度,而Y轴表示相应荧光强度的细胞计数。

  1. 重叠阳性对照的DiOC6(3)信号,表明线粒体膜电位(MMP)的消耗量。


    图3.健康细胞的重叠直方图(红色)和线粒体膜电位的细胞损失(灰色)。健康细胞的D 2 O 6(3)直方图和MMP的细胞损失重叠,以比较强度 差异。

  1. 控制MMP(R1)和健康细胞(R2)的细胞损失

    图4.选择健康(R2)或贫化MMP(R1)的细胞群体。在R1中门控具有高强度的DiOC6(3)的健康细胞, 在R1中门控具有较低DiOC6(3)强度的MMP的细胞损失。

食谱

  1. DPBS(1 L)
    8克氯化钠(NaCl)
    0.2克氯化钾(KCl)
    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. DiOC 6原料(10mM)
    将10 mg溶于1.747 ml DMSO中,制成1 mM储备液。
    等分并储存在-20°C 避免光照和反复冻融。
  3. 多聚甲醛原料(8%)
    重量8g多聚甲醛加到90ml蒸馏水中(在通风橱中) 加入0.1ml 10N NaOH 加热并搅拌溶液直至颗粒完全溶解(不要在65℃以上加热溶液) 关闭加热器,用约0.3ml的20%HCl调节至pH7.4 用蒸馏水将体积调至100ml。
    用0.22μm过滤器消毒溶液,并可在4℃下储存30-60天

参考文献

  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 Mitochondrial Membrane Potential. Bio-protocol 3(8): e430. DOI: 10.21769/BioProtoc.430.
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Rodrigo Hoyos
Boston Children's Hospital
Hi, I've been using this protocol in fibroblasts, but even thou the staining works I don't get a depletion of the mitochondrial membrane potential in the cells fixed with fresh 4% paraformaldehyde. If I compare the MFI of both the fixed and non fixed cells, the MFI is higher in the fixed ones, do you have any suggestions on how to solve this? Since the fixation did not work properly I analyzed the membrane potential comparing live and dead cells without fixation, but I'm not sure that's a proper control for the loss of membrane potential. I will be thankful for any help you could give me.

many thanks

Rodrigo
12/2/2015 2:09:36 PM Reply
Hsin-Yi Chang
Kyoto University

Hi Rodrigo,

Positive charged fluorescent dyes such as DiOC6 accumulates in mitochondria due to their large negative membrane potential. Therefore, the dye can not be retained in mitochondria after incubation of any reagent causes membrane permeabilization, e.g. aldehyde fixation or detergent permeabilization. Alternatively, you can treat cells with mitochondrial uncoupling agents for mitochondrial depolarization. We've routinely used the paraformaldehyde as the positive control for membrane potential depletion and works well. You can check whether you washed the cells thoroughly after paraformaldehyde treatment and DiOC6 staining, and used the same cell number for staining.

Hsin-Yi

12/2/2015 5:23:34 PM