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Detection of Membrane Potential in Mycobacterium tuberculosis
检测结核杆菌中的膜电位   

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Abstract

DiOC2 (Novo et al., 2000) exhibits green fluorescence in all bacterial cells, but the fluorescence shifts towards red emission as the dye molecules self associate at the higher cytosolic concentrations caused by larger membrane potentials. Proton ionophores such as CCCP destroy membrane potential by eliminating the proton gradient. The magnitude of membrane potentials varies with different bacterial species. For many gram-positive species, including Staphylococcus aureus and Micrococcus luteus, the red:green ratio tends to vary with the intensity of the proton gradient while in many gram-negative bacteria such as Escherichia coli and Salmonella choleraesuis, the response of the dye does not appear to be proportional to proton gradient intensity. Mycobacterium tuberculosis itself is a difficult organism to work with because of its rigid cell wall.

Materials and Reagents

  1. Mycobacterial cells (Mycobacterium tuberculosis)
  2. 7H9 broth (Difco, catalog number: 271310 )
  3. Glycerol (Amresco, catalog number: 0854 )
  4. TWEEN®80 (Amresco, catalog number: 0442 )
  5. Middlebrook ADC (albumin-dextrose-catalase) enrichment (BD Biosciences, catalog number: 211886 )
  6. The BacLightTM Bacterial Membrane Potential Kit (Invitrogen, catalog number: B34950 )
    Provides the following solution:
    1. DiOC2 (Component A), 1.2 ml of a 3 mM solution in DMSO
    2. CCCP (Component B), 300 μl of a 500 μM solution in DMSO
    3. Phosphate-buffered saline (PBS, Component C), 10 mM sodium phosphate and 145 mM sodium chloride, pH 7.4
  7. Supplenmented 7H9 broth (see Recipes)

Equipment

  1. Incubation shaker
  2. Laminar flow hood
  3. Laboratory centrifuge
  4. Flow cytometer (BD FACSVerseTM System)

Procedure

  1. Culture the mycobacterial cells aerobically in 7H9 broth at 37 °C with shaking (200 rpm) till mid- log phase (OD600 of 0.3).
  2. Filter the required volume of PBS (Component C) through a 0.22 μm pore size membrane, preparing enough for culture dilution and 500 μl per test.
  3. Allow the 3 mM DiOC2 (Novo et al., 2000) and 500 μM CCCP solutions (Components A and B) to come to room temperature before use.
  4. Wash the cells with 1 ml filtered PBS twice. Spin down at a speed of 4,000 rpm for 5 min for each wash.
  5. Dilute the mycobacterial culture to approximately 1 x 106 cells per ml in filtered PBS.
  6. Aliquot 500 μl of the bacterial suspension into a flow cytometry tube for each staining experiment to be performed. Prepare two additional tubes for a depolarized control and an unstained control.
  7. Add 25 μl of 500 μM CCCP (Component B) to the depolarized control sample and mix.
  8. Add 3 μl of 3 mM DiOC2 Component A) to each flow cytometry tube and mix (do not add stain to the unstained control sample). Incubate samples at room temperature for 30 min. Stained samples can be analyzed after 5 min, but signal intensity continues to increase until about 30 min.
  9. Stained bacteria can be assayed in a flow cytometer equipped with a laser emitting at 488 nm. Fluorescence is collected in the green and red channels; filters used for detecting fluorescein and the Texas Red dye, respectively, are generally suitable. The forward scatter, side scatter, and fluorescence should be collected with logarithmic signal amplification.
  10. Instrument adjustments are especially critical for detecting relatively small particles such as bacteria. Use the unstained control sample to locate bacterial populations in the forward and side scatter channels. Use the side scatter as the parameter for setting the acquisition trigger.
  11. After adjusting the flow cytometer as described above, apply the depolarized control sample. Gate on bacteria using forward versus side scatter and adjust fluorescence photomultiplier tube voltages such that the green and red MFI values are approximately equal. Do not set compensation.
  12. While the relative amount of red and green fluorescence intensity will vary with cell size and aggregation, the ratio of red to green fluorescence intensity can be used as a size-independent indicator of membrane potential. The data can also be processed by gating on bacteria using forward versus side scatter, and analyze gated populations with a dot plot of red versus green fluorescence reporting MFI values as linear values, not as channels.
  13. On a ratiometric histogram, set markers around the peaks of interest and record the mean ratio values (Figure 1). For a dot plot of red versus green fluorescence, set regions around the populations of interest and record red and green MFI values for each. To evaluate the data, divide the red population MFI by the green population MFI.
  14. In the flow cytometer, bacteria are identified solely on the basis of their size and stain ability.


    Figure 1. Detection of membrane potential in mycobacteria. Red/green ratios were calculated using population mean fluorescence intensities for mycobacteria, incubated with 3 μM DiOC2 for 30 min in either the presence or absence of 25 μM CCCP.

Notes

  1. Carbocyanine dyes, including DiOC2 and CCCP, are inhibitors of respiration. While these dyes do not alter assay results over the recommended staining periods, both DiOC2 and CCCP are toxic to bacterial cells and the cells will not be culturable after even brief exposure.
  2. The cells should be washed properly and any kind of aggregation should be avoided as it might interfere with the assay. In case any aggregates are still observed, allow the tubes to stand for a while so that the clumps can settle down. The sample can be transferred to a fresh tube.
  3. Solutions of the carbocyanine dye DiOC2 (3,3′-diethyloxacarbocyanine iodide) and CCCP (carbonyl cyanide 3-chlorophenylhydrazone) should be protected from light.

Recipes

  1. 7H9 broth supplemented with 0.1% glycerol, 0.1% TWEEN®80 and Middlebrook ADC (albumin-dextrose-catalase) enrichment

Acknowledgments

Dr. Amit Singh is a Wellcome- DBT India Alliance Intermediate Fellow. The work was supported by the Wellcome- DBT India Alliance grant, WTA01/10/355.

References

  1. Chawla, M., Parikh, P., Saxena, A., Munshi, M., Mehta, M., Mai, D., Srivastava, A. K., Narasimhulu, K. V., Redding, K. E., Vashi, N., Kumar, D., Steyn, A. J. and Singh, A. (2012). Mycobacterium tuberculosis WhiB4 regulates oxidative stress response to modulate survival and dissemination in vivo. Mol Microbiol 85(6): 1148-1165.
  2. Novo, D. J., Perlmutter, N. G., Hunt, R. H. and Shapiro, H. M. (2000). Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus. Antimicrob Agents Chemother 44(4): 827-834.
  3. Novo, D., Perlmutter, N. G., Hunt, R. H. and Shapiro, H. M. (1999). Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique. Cytometry 35(1): 55-63.

简介

DiOC 2(Novo等人,2000)在所有细菌细胞中表现出绿色荧光,但是随着染料分子在更高的胞质浓度自缔合,荧光向红色发射移动 由较大的膜电位引起。 质子离子载体如CCCP通过消除质子梯度来破坏膜电位。 膜电位的大小随不同的细菌种类而变化。 对于许多革兰氏阳性菌种,包括金黄色葡萄球菌和藤黄微球菌,红色:绿色比倾向于随着质子梯度的强度而变化,而在许多革兰氏阴性菌中 例如大肠杆菌和霍乱沙门氏菌,染料的响应似乎不与质子梯度强度成比例。 结核分枝杆菌本身是一种难以处理的生物体,因为其刚性细胞壁。


材料和试剂

  1. 分枝杆菌细胞(结核分枝杆菌)
  2. 7H9肉汤(Difco,目录号:271310)
  3. 甘油(Amresco,目录号:0854)
  4. (Amresco,目录号:0442)
  5. Middlebrook ADC(白蛋白 - 葡萄糖 - 过氧化氢酶)富集(BD Biosciences,目录号:211886)
  6. Light 细菌膜电位试剂盒(Invitrogen,目录号:B34950)
    提供以下解决方案:
    1. DiOC 2(组分A),1.2ml 3mM的DMSO溶液
    2. CCCP(组分B),300μl500μM的DMSO溶液
    3. 磷酸盐缓冲盐水(PBS,组分C),10mM磷酸钠和145mM氯化钠,pH 7.4
  7. 补充7H9肉汤(见配方)

设备

  1. 孵育振动器
  2. 层流罩
  3. 实验室离心机
  4. 流式细胞仪(BD FACSVerseTM系统)

程序

  1. 在37℃下,在振荡(200rpm)下,在7H9肉汤中有氧地培养分枝杆菌细胞直到中对数期(OD 600为0.3)。
  2. 通过0.22μm孔径的膜过滤所需体积的PBS(组分C),准备足够的培养稀释液,每次测试500μl。
  3. 在使用前允许3mM DiOC 2(Novo等人,2000)和500μMCCCP溶液(组分A和B)达到室温。< br/>
  4. 用1ml过滤的PBS洗涤细胞两次。 每次洗涤以4,000rpm的速度旋转5分钟。
  5. 在过滤的PBS中将分枝杆菌培养物稀释至约1×10 6个细胞/ml。
  6. 将500微升细菌悬浮液分装到流式细胞仪管中进行每次染色实验。 准备两个额外的管用于去极化对照和未染色对照
  7. 向去极化的对照样品中加入25μl500μMCCCP(组分B)并混合
  8. 向每个流式细胞仪管中加入3μl3mM DiOC 2成分A)并混合(不向未染色的对照样品中加入染色剂)。在室温下孵育样品30分钟。染色的样品可以在5分钟后分析,但信号强度继续增加直到约30分钟
  9. 染色的细菌可以在配备有在488nm发射的激光的流式细胞仪中测定。荧光收集在绿色和红色通道中;用于分别检测荧光素和德克萨斯红染料的滤光片通常是合适的。前向散射,侧向散射和荧光应该用对数信号放大来收集
  10. 仪器调节对于检测相对小的颗粒例如细菌是特别关键的。使用未染色的对照样品来定位前向和侧向散射通道中的细菌群体。使用侧散射作为设置采集触发的参数。
  11. 在如上所述调节流式细胞仪之后,应用去极化的对照样品。使用正向对侧向散射和调整荧光光电倍增管电压使得绿色和红色MFI值近似相等的细菌门。不要设置补偿。
  12. 虽然红色和绿色荧光强度的相对量将随细胞大小和聚集而变化,但是红色与绿色荧光强度的比率可以用作膜电位的大小无关指示物。还可以通过使用正向对侧散射对细菌进行门控来处理数据,并且使用红色与绿色荧光的点图分析门控群体,将MFI值报告为线性值而不是通道。
  13. 在比率直方图上,在感兴趣的峰周围设置标记,并记录平均比值(图1)。对于红色与绿色荧光的点图,设置感兴趣群体周围的区域,并记录每个的红色和绿色MFI值。为了评估数据,将红色群体MFI除以绿色群体MFI。
  14. 在流式细胞仪中,细菌仅根据其大小和染色能力来鉴定

    图1.分枝杆菌中的膜电位的检测使用分枝杆菌的群体平均荧光强度计算红/绿比率,所述分枝杆菌与3μMDiOC 2 2孵育30分钟,存在或不存在25μMCCCP

笔记

  1. 碳菁染料,包括DiOC 2和CCCP,是呼吸的抑制剂。 虽然这些染料在推荐的染色期间不改变测定结果,但是DiOC 2和CCCP对细菌细胞都是有毒的,并且即使短暂的暴露也不能培养细胞。
  2. 细胞应该被适当地洗涤并且应该避免任何种类的聚集,因为它可能干扰测定。 在仍然观察到任何聚集体的情况下,允许管静置一段时间,使得团块可以沉降。 样品可以转移到新管中
  3. 应该保护碳菁染料DiOC 2(碘化3,3'-二乙基氧杂羰花青)和CCCP(羰基氰化物3-氯苯基腙)的溶液不受光照。

食谱

  1. 7H9肉汤,补充有0.1%甘油,0.1%80和Middlebrook ADC(白蛋白 - 葡萄糖 - 过氧化氢酶)富集

致谢

Amit Singh博士是一个Wellcome-DBT印度联盟中级研究员。 这项工作得到了Wellcome-DBT印度联盟资助WTA01/10/355的支持。

参考文献

  1. Chawla,M.,Parikh,P.,Saxena,A.,Munshi,M.,Mehta,M.,Mai,D.,Srivastava,AK,Narasimhulu,KV,Redding,KE,Vashi,N.,Kumar,D 。,Steyn,AJand Singh,A.(2012)。 结核分枝杆菌 em> WhiB4调节氧化应激反应以调节体内存活和传播。
  2. Novo,D.J.,Perlmutter,N.G.,Hunt,R.H。和Shapiro,H.M。(2000)。 多参数流式细胞术分析抗生素对膜电位,膜通透性和细菌计数的影响 金黄色葡萄球菌和微球菌。 抗微生物剂化学 44(4):827-834。
  3. Novo,D.,Perlmutter,N.G.,Hunt,R.H。和Shapiro,H.M。(1999)。 使用二乙基氧羰花青的细菌中精确的流式细胞膜电位测量和比例计算技术 Cytometry 35(1):55-63
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chawla, M. and Singh, A. (2013). Detection of Membrane Potential in Mycobacterium tuberculosis . Bio-protocol 3(11): e785. DOI: 10.21769/BioProtoc.785.
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Melody Agre
ASU
When using the BacLightTM Bacterial Membrane Potential Kit, will it only stain the bacterium in a bacterium-host cell interaction?
5/20/2015 12:45:58 PM Reply
Amit Singh
Immunology Department, International Centre for Genetic Engineering and Biotechnology, India

Since this kit is specific for bacterial cells, the host cells will not get stained.

5/22/2015 5:00:43 AM


sudipa galgalkar
siemens
Can we carryout this experiment with Fluorescent microscope? If so then which excitation/emission wavelength should i select
2/26/2014 1:16:03 AM Reply
Amit Singh
Immunology Department, International Centre for Genetic Engineering and Biotechnology, India

Yes this experiment can be carried out with fluorescent microscopy. The green fluorescence of DIOC2(3) requires 488 nm excitation and 530 nm emission while the red fluorescence requires 488 nm excitation and 610 nm emission. Hope that will help!

3/14/2014 1:58:02 AM


sudipa galgalkar
siemens

Many Thanks Manbeena!!

3/17/2014 9:18:28 PM