搜索

Lysosomal Stability Assay
溶酶体稳定性分析   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Cancer Cell
Sep 2013

Abstract

This assay makes use of the dye Acridine Orange (AO) to determine the stability of lysosomes in living cells upon exposure to a confocal microscope laser.

AO is a lipophilic amine that readily diffuses into cells (Figure 1). Inside the cell it enters the acidic lysosomal compartment where it is protonated and sequestered, shifting its emission spectrum towards a longer wavelength (i.e. red). Once inside the lysosomes, the metachromatic AO sensitizes the lysosomal membrane to photo-oxidation by blue light (Brunk et al., 1997). Upon light-induced loss of the lysosomal pH gradient and subsequent leakage of AO into the cytosol, the emission spectrum of AO shifts from red to green (Figure 2). Hence, loss of lysosomal integrity can be measured as a ‘loss of red dots’ or as a quantitative rise in green fluorescence (Petersen et al., 2010; Kirkegaard et al., 2010; Petersen et al., 2013).


Figure 1. Acridine Orange


Figure 2. Snapshots visualizing the U2OS cells at various steps of the recording procedure (Petersen et al., 2010)

Materials and Reagents

  1. Cells (such as U-2 OS, ATCC, catalog number: HTB-96)
  2. Growth medium (such as RPMI 1640 with 6% serum, Life Technologies, Gibco®)
  3. Acridine Orange (Sigma-Aldrich, catalog number: 235474)
  4. PBS containing 3% serum

Equipment

  1. Zeiss Live DUO 5 confocal microscope or any newer microscope model capable of recording confocal images at high speed (500 ms/exposure), 100 mW diode laser, 40x c-apochromat objective
  2. Lab-Tek 4 well (Nunc®, catalog number: 155383 ) or 8 well (Nunc®, catalog number: 155411 ) borosilicate cell chambers

Procedure

Notes on protocol versions: This protocol exists in two versions, one for use with common cancer cell lines (such as U2OS or Mcf7) and one for mouse embryonic fibroblasts (MEFs) transduced with either the SrcY527F oncogene or empty vector (or indeed any other transduced genes of interest). The main difference between the two protocols is in the recording of the images on the confocal microscope. When needed, both versions will be described at each step and designated either COMMON or MEF.

  1. Grow cells in borosilicate lab-tek wells overnight or for as long as required by necessary treatments. The exact nature of the growth medium is not important. The amount of cells in each well on the day of analysis is critical to the successful use of this protocol. In order to keep the variation low, the cells should be 50-75% confluent, and should never reach 100% confluency.
  2. Add Acridine Orange directly to the growth medium to a final concentration of 2 µg/ml. Incubate the cells at 37 ºC for 20 min.
  3. Wash the cells twice in PBS containing 3% serum.
  4. Add PBS with 3% serum to cover the cells (100 or 200 µl for 8-well or 4-well respectively).
  5. Proceed immediately to analysis at the microscope. The cells are only suited for analysis for the first 60 min after washing. After this time, the variation between different observation fields will increase.
  6. Identify a group of cells in the brightfield, and make sure the lysosomes (visible as black dots) are in focus. Be quick at this point, since brightfield also contains blue light.
  7. Microscope settings:
    This protocol was developed and used on a Zeiss LSM 5 LIVE DUO microscope. It is important to note that, as for all microscope protocols, the exact settings should be determined empirically for each instrument and experimental setup. As noted above, the protocol used for ‘normal’ cancer cell lines (i.e. U2OS & Mcf7) was different from the one used for MEFs. The slower protocol with less frames (and less exposure to blue light) for MEFs was developed since lysosomes loose their integrity a lot faster in these cells, making quantification challenging when recorded at high speed.
    Heat chamber
    37 ºC
    Ocular
    40x c-apochromat
    Laser intensity
    2.6 (most cancer cell lines)/0.9 (MEF) (can vary greatly between instruments)
    Image quality
    8 bit (16 bit makes the files unnecessary large)
    Recording speed
    1 frame every 0.5 (most cancer cell lines)/12 sec (MEF)
    Images recorded
    200 (most cancer cell lines)/120 (MEF)
    Laser wavelength
    489 nm
    Recording filters
    495-555 nm band pass (green), 650 nm long pass (red)
  8. Record movies. Any focus corrections should be made within the first 10 sec of recording. Make sure never to record the same place twice (easy to see due to absence of red dots). Allow time for all wells to be analyzed within an hour. Note the order in which the recordings were made, for use in troubleshooting later.

Data Analysis

The data was analyzed using the LSM5 DUO software. This software has since been upgraded by Zeiss to various versions of ZEN. Unfortunately a key functionality for this analysis was omitted in this update, so data analysis was performed in the older software. Hopefully this will be rectified by Zeiss, and maybe other microscope and software suppliers don’t have these issues.

  1. Open the movie in the Zeiss software.
  2. Choose ROI (region of interest) analysis.
  3. Determine the minimum pixel intensity to be analyzed for each channel (or colour). This is done using the “level” function. At the time of writing, the ZEN software could only do this on single images, not the multiple image frames making up the movies recorded in this protocol. The older Zeiss software can do this.
    1. Forward the movie to the first frame after 10 sec.
    2. Mark the entire frame with the ROI tool. In previous versions of this protocol individual cells were marked manually, however this was very labour intensive, and will not produce better results.
    3. For the green channel, change the minimum level of pixel intensity to be analyzed, so that all background is omitted (The entire area of cells should be visible with a weak green stain.).
    4. For the red channel, change the level so that only brightly red stained lysosomes are analyzed.
    5. These same numeric settings should be used for all movies.
    6. Since each frame contains multiple cells, the values calculated by the software should now reflect the average green and red intensity of the cells. Observe that the green values increase while the red values decrease as the lysosomes burst.
    7. Copy the values from the microscope software directly to Excel or a similar program.
  4. In excel, line up data sets that come from identical experimental settings. At least three independent experiments, each with 3-4 movies, should be analyzed.
  5. Normalize each movie to its value at 10 sec.
  6. Visualize the increase in green in a x/y scatter plot. The decrease in red is much less reliable than the increase in green. Warning, this produces large and complex spreadsheets (Figure 3).
  7. For statistical evaluation, a simple students t-test can be performed for individual time points (for example the control value at 20 sec versus the treated value at 20 sec).


    Figure 3. Example of data set in Excel

Notes

It is important to emphasize that great care should be taken to standardize the cell growth conditions, as the outcome of this assay is greatly affected by confluency and the general health status of the cells. Also, lipid mediated transfection protocols can influence the integrity of lysosomes negatively, making them more unstable. Finally, it is expected that at the end of each movie, the intensity of the green staining will start to decrease towards the end of each movie, so when showing graphs it is often required to omit the late values for clarity.

Acknowledgments

This protocol was developed for two papers addressing the stability of lysosomes (Petersen et al., 2013 and Kirkegaard et al., 2010). The work was supported by grants from the Danish Cancer Society (to NHTP and MJ), and the Danish National Research Foundation, the Danish Council for Independent Research, the European Commission FP7 and the Novo Nordisk Foundation (to MJ).

References

  1. Brunk, U. T., Dalen, H., Roberg, K. and Hellquist, H. B. (1997). Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts. Free Radic Biol Med 23(4): 616-626.
  2. Kirkegaard, T., Roth, A. G., Petersen, N. H., Mahalka, A. K., Olsen, O. D., Moilanen, I., Zylicz, A., Knudsen, J., Sandhoff, K., Arenz, C., Kinnunen, P. K., Nylandsted, J. and Jäättelä, M. (2010). Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology. Nature 463(7280): 549-553.
  3. Petersen, N. H., Kirkegaard, T., Olsen, O. D. and Jäättelä, M. (2010). Connecting Hsp70, sphingolipid metabolism and lysosomal stability. Cell Cycle 9(12): 2305-2309.
  4. Petersen, N. H., Olsen, O. D., Groth-Pedersen, L., Ellegaard, A. M., Bilgin, M., Redmer, S., Ostenfeld, M. S., Ulanet, D., Dovmark, T. H., Lonborg, A., Vindelov, S. D., Hanahan, D., Arenz, C., Ejsing, C. S., Kirkegaard, T., Rohde, M., Nylandsted, J. and Jäättelä, M. (2013). Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase. Cancer Cell 24(3): 379-393.

简介

该测定利用染料吖啶橙(AO)确定在暴露于共焦显微镜激光器时活细胞中溶酶体的稳定性。


图1。吖啶橙

AO是一种易于扩散入细胞的亲脂胺(图1)。在细胞内部,它进入酸性溶酶体区室,在那里它被质子化和螯合,将其发射光谱移向更长的波长(即,红色)。一旦在溶酶体内,异染色质AO使溶酶体膜通过蓝光的光氧化敏感(Brunk等人,1997)。在光诱导的溶酶体pH梯度的损失和随后的AO向胞质溶胶中的泄漏后,AO的发射光谱从红色变为绿色(图2)。因此,溶酶体完整性的损失可以被测量为"红点的丢失"或作为绿色荧光的定量升高(Petersen等人,2010; Kirkegaard等人,,2010; Petersen ,2013)。


图2.快照在记录过程的不同步骤(Petersen ,2010)下可视化U2OS细胞

材料和试剂

  1. 细胞(例如U-2OS,ATCC,目录号:HTB-96)
  2. 生长培养基(例如具有6%血清的RPMI 1640,Life Technologies,Gibco )
  3. 吖啶橙(Sigma-Aldrich,目录号:235474)
  4. 含3%血清的PBS

设备

  1. Zeiss Live DUO 5共聚焦显微镜或任何能够以高速(500ms /曝光),100mW二极管激光器,40x c-消色差物镜物镜记录共焦图像的新型显微镜型号
  2. Lab-Tek 4孔(目录号:155383)或8孔(目录号:155411)的硼硅酸盐电池室

程序

协议版本的注意事项:此协议存在两个版本,一个用于常见的癌细胞系(如U2OS或Mcf7)和一个用于转导SrcY527F癌基因或空载体的小鼠胚胎成纤维细胞(MEF)(或 实际上任何其他感兴趣的转导的基因)。 两种方案之间的主要区别是在共焦显微镜上记录图像。 当需要时,将在每个步骤和两个版本进行描述 指定为COMMON或MEF。

  1. 在硼硅酸盐实验室孔中生长细胞过夜或根据必要的治疗所需时间。生长培养基的确切性质并不重要。在分析当天,每个孔中的细胞量对于该方案的成功使用是关键的。为了保持变化低,细胞应该是50-75%汇合,并且不应该达到100%汇合。
  2. 将吖啶橙直接加入到生长培养基中至终浓度为2μg/ml。在37℃孵育细胞20分钟。
  3. 在含有3%血清的PBS中洗涤细胞两次
  4. 加入含3%血清的PBS以覆盖细胞(分别为8孔或4孔的100或200μl)。
  5. 立即进行在显微镜下的分析。细胞仅适用于洗涤后第一个60分钟的分析。此后,不同观察视野之间的变化将增加。
  6. 识别明亮的领域中的一组细胞,并确保溶酶体(可视为黑点)在焦点。在这一点很快,因为brightfield也包含蓝光。
  7. 显微镜设置:
    该方案开发并用于Zeiss LSM 5LIVE DUO显微镜。重要的是要注意,对于所有的显微镜协议,确切的设置应该根据经验确定每个仪器和实验设置。如上所述,用于"正常"癌细胞系(即U2OS和Mcf7)的方案不同于用于MEF的方案。由于溶酶体在这些细胞中快速失去它们的完整性,使得定量在高速记录时具有挑战性,因此开发了用于MEF的较少帧(和较少暴露于蓝光)的较慢方案。
    加热室
    37ºC
    眼睛
    40x c-apochromat
    激光强度
    2.6(大多数癌细胞系)/0.9(MEF)(仪器之间差异很大)
    图像质量
    8位(16位使文件不必要大)
    录制速度
    1帧每0.5(大多数癌细胞系)/12秒(MEF)
    图片已记录
    200(大多数癌细胞系)/120(MEF)
    激光波长
    489 nm
    录制过滤器
    495-555nm带通(绿色),650nm长通(红色)
  8. 录制电影。 任何焦点校正应在记录的前10秒内进行。 确保不要记录相同的地方两次(容易看到,由于没有红点)。 允许在一小时内分析所有孔的时间。 请注意记录的顺序,以便稍后进行故障排除。

数据分析

使用LSM5 DUO软件分析数据。 这个软件已经被蔡司升级到各种版本的ZEN。 不幸的是,此更新中省略了此分析的关键功能,因此在旧版软件中执行数据分析。 希望这将由Zeiss纠正,也许其他显微镜和软件供应商没有这些问题。

  1. 在Zeiss软件中打开电影。
  2. 选择ROI(感兴趣区域)分析。
  3. 确定每个通道(或颜色)要分析的最小像素强度。 这是使用"level"函数完成的。 在写作时,ZEN软件只能在单个图像上执行此操作,而不是构成此协议中记录的电影的多个图像帧。 较早的Zeiss软件可以做到这一点。
    1. 10秒后将电影转发到第一帧。
    2. 标记 整个框架与ROI工具。 在此协议的以前版本 单个细胞被手动标记,然而这是非常劳动 密集,不会产生更好的效果。
    3. 为绿色 通道,改变要分析的像素强度的最小水平,所以 所有背景都省略(单元格的整个区域应该是 用弱绿色染色可见)。
    4. 对于红色通道,更改水平,以便只分析亮红色的溶酶体
    5. 所有电影都应使用相同的数字设置。
    6. 由于每个帧包含多个单元格,由   软件现在应该反映平均绿色和红色强度 细胞。 观察到绿色值增加而红色值 随溶酶体爆发而减少
    7. 将值从显微镜软件直接复制到Excel或类似的程序。
  4. 在excel中,排列来自相同实验设置的数据集。 应分析至少三个独立实验,每个实验有3-4部电影
  5. 将每部电影标准化为其值为10秒。
  6. 在x/y散点图中可视化绿色的增加。 红色的减少远不如绿色的增加可靠。 警告,这会产生大而复杂的电子表格(图3)。
  7. 对于统计评估,可以对各个时间点(例如,20秒时的对照值与20秒时的治疗值)进行简单的学生t检验。


    图3. Excel中的数据集示例

笔记

重要的是要强调,应该非常小心地标准化细胞生长条件,因为该测定的结果受到融合和细胞的一般健康状态的极大影响。此外,脂质介导的转染方案可以负面影响溶酶体的完整性,使它们更不稳定。最后,预计在每部电影结束时,绿色染色的强度将朝向每个电影的结束开始减小,因此当显示图形时,为了清楚起见通常需要省略晚期值。

致谢

该方案是针对解决溶酶体稳定性的两篇论文开发的(Petersen等人,2013和Kirkegaard等人,2010)。这项工作得到了丹麦癌症协会(NHTP和MJ),丹麦国家研究基金会,丹麦独立研究委员会,欧洲委员会FP7和Novo Nordisk基金会(MJ)的资助。

参考文献

  1. Brunk,U.T.,Dalen,H.,Roberg,K。和Hellquist,H.B。(1997)。 溶酶体膜的光氧化破坏导致培养的人成纤维细胞凋亡。 Free Radic Biol Med 23(4):616-626。
  2. Kirkengaard,T.,Roth,AG,Petersen,NH,Mahalka,AK,Olsen,OD,Moilanen,I.,Zylicz,A.,Knudsen,J.,Sandhoff,K.,Arenz,C.,Kinnunen, Nylandsted,J。和Jäättelä,M。(2010)。 Hsp70可稳定溶酶体并恢复尼曼 - 皮克病相关溶酶体病理。 自然 463(7280):549-553。
  3. Petersen,N.H.,Kirkegaard,T.,Olsen,O.D.andJäättelä,M。(2010)。 连接Hsp70,鞘脂代谢和溶酶体稳定性 细胞周期 9(12):2305-2309。
  4. Petersen,NH,Olsen,OD,Groth-Pedersen,L.,Ellegaard,AM,Bilgin,M.,Redmer,S.,Ostenfeld,MS,Ulanet,D.,Dovmark,TH,Lonborg,A.,Vindelov,SD ,Hanahan,D.,Arenz,C.,Ejsing,CS,Kirkegaard,T.,Rohde,M.,Nylandsted,J.andJäättelä,M.(2013)。 神经鞘脂代谢的转化相关变化使细胞对由酸性鞘磷脂酶抑制剂诱导的溶酶体细胞死亡敏感。/a> Cancer Cell 24(3):379-393。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Petersen, N. H., Kirkegaard, T. and Jäättelä, M. (2014). Lysosomal Stability Assay. Bio-protocol 4(12): e1162. DOI: 10.21769/BioProtoc.1162.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。