搜索

Mitochondrial Biogenesis Assay after 5-day Treatment in PC-3 Cells
在PC-3细胞中处理5天后的线粒体生成分析

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Drug-induced mitochondrial injury can be caused by many different mechanisms including inhibition of mitochondrial DNA replication, transcription, translation, and altered protein function. Determination of the level of mitochondrial protein synthesis, or mitochondrial biogenesis, relative to the cellular protein synthesis, provides important information on potential mitochondrial toxicity.

Keywords: Mitochondrial toxicity(线粒体毒性), Mitochondrial protein synthesis(线粒体蛋白质合成), Nucleoside-related toxicity(核苷类药物相关毒性), Chloramphenicol(氯霉素), MitoBiogenesis™ In-Cell ELISA Kit(mitobiogenesis™细胞ELISA Kit)

Materials and Reagents

  1. PC-3 cells (ATCC, CRL-1435, catalog number: 7348669 )
  2. Three-fold serial dilutions of compounds
  3. MitoToxTM MitoBiogenesisTM In-Cell ELISA Kit (Abcam, catalog number: ab110216 ) including
    1. 1,000x IRDye®-labeled secondary antibodies (species were not disclosed by vendor)
    2. 200x primary antibodies (species were not disclosed by vendor)
    3. 100x Triton X-100
    4. 10x blocking buffer
    5. 10x phosphate buffered saline (PBS)
    6. 400x Tween-20
    7. 1x Janus Green stain (optional)
  4. DMSO (cell culture grade) (Sigma-Aldrich, catalog number: D2650 )
  5. CellTiter Glo (Promega Corporation, catalog number: G7571 )
  6. Kaighn’s F12 (Life Technologies, Gibco®, catalog number: 21127 )
  7. FBS (HyClone, catalog number: SH30071.03 )
  8. 100 units/ml penicillin and 100 µg/ml streptomycin (P/S) (Life Technologies, Gibco®, catalog number: 15140-122 )
  9. Cell fixing reagent: 4% paraformaldehyde (20% paraformaldehyde, VWR International, catalog number: 15713-S )
  10. Chloramphenicol (10 mM solution in DMSO) (Sigma-Aldrich, catalog number: C1919-5G )
  11. Kaighn’s F12 medium (see Recipes)
    Note: The cells were maintained in Kaighn’s F12 medium. The cells were passaged twice weekly and maintained at 90% confluence. Cells between passages 4 and 15 were used in the assays.

Equipment

  1. 96-well plates (collagen I coated 96-well plate) (VWR International, catalog number: 73521-028 )
  2. Titer plate shaker (Lab-line Instrument, model: 4626 )
  3. LI-COR® Odyssey instrument (LI-COR)

Software

  1. GraphPad Prism 5.0 (GraphPad Software)

Procedure

  1. PC-3 cells were plated at a density of 2.5 x 103 cells per well in a final assay volume of 100 μl per well in a collagen coated 96-well plate one day before the treatment.
  2. On the treatment day, three-fold serial dilutions of compounds in DMSO were prepared in duplicate in 96-well plates starting at a concentration close to the general cytotoxicity 5-day CC50 value of the compound. The CC50 value was measured by monitoring changes in the cellular ATP level. For compounds with general cytotoxicity CC50 value ≥ 100 μM, the starting concentration was at 100 μM. A representative 96-well plate map is shown in Figure 1.
    1. Outer-wells, labeled as Blank in Figure 1, were treated with 0.5% DMSO and used as a background for future data analysis.
    2. 0.5% DMSO treated cells, labeled as DMSO control in Figure 1, were used as a non-drug treated control.
  3. Compounds were mixed with medium to make 2x compound-medium solution, and then 100 µl/well of 2x compound-medium solution was added to the relevant wells in a 96-well cell plate. The final volume of the well was 200 µl. The final DMSO content was 0.5%.
  4. After 5-day incubation, the cells were fixed with 50 µl/well of 4% paraformaldehyde for 20 minutes at room temperature. After fixing, the cells were washed with 1x PBS twice (prepared from dilution of 10x PBS).
    1. At this point the plate could be stored at 4 °C up to a week with 100 µl/well of 1x PBS.
  5. The cells were analyzed with the MitoBiogenesisTM In-Cell ELISA Kit, which uses quantitative immunocytochemistry to measure protein levels of Complexes II (SDH-A) and IV (COX-1) in cultured cells, which involved the following steps:
    1. The cells were permeabilized with 100 µl/well of 1x permeabilization buffer (prepared from dilution of 100x Triton X-100 with 1x PBS) for 30 min at room temperature with gentle agitation on a titer plate shaker at constant speed (setting 2 on a Lab-line Instrument). The same speed setting was used in all of the following steps when a shaker was used.
    2. After removing the permeabilization buffer from the cell plate, the cells were incubated with 2x blocking buffer (200 µl/well) for 2 h at room temperature with gentle agitation on a titer plate shaker.
      1. 2x blocking buffer was prepared from dilution of 10x blocking buffer with 1x PBS.
    3. After removing the blocking buffer, the cells were incubated with 1x primary antibodies in 1x blocking buffer (100 µl/well) overnight at 4 °C with gentle agitation on a titer plate shaker.
      1. 1x blocking buffer was prepared from dilution of 10x blocking buffer with 1x PBS.
      2. 1x primary antibodies was prepared from dilution of 200x primary antibodies with 1x blocking buffer.
      3. The outer-wells were treated with 1x blocking buffer without antibodies.
    4. On the following day, the cells were washed three times with 1x PBS-Tween buffer (300 µl/well, 3 times).
      1. 1x PBS-Tween buffer was prepared from dilution of 400x Tween-20 with 1x PBS.
    5. After washing, the cells were incubated with 1x IRDye®-labeled secondary antibody in 1x blocking buffer (100 µl/well) for 2 h at room temperature with gentle agitation on a titer plate shaker.
      1. 1x blocking buffer was prepared from dilution of 10x blocking buffer with 1x PBS.
      2. 1x IRDye®-labeled secondary antibodiys was prepared from dilution of 1,000x IRDye®-labeled secondary antibodies with 1x blocking buffer.
      3. The outer-wells were treated with 1x blocking buffer without antibodies.
    6. The cells were washed with 1x PBS-Tween buffer (300 µl/well) for 5 times and kept in 1x PBS-Tween buffer (200 µl/well) till imaging.
      1. 1x PBS-Tween buffer was prepared from dilution of 400x Tween-20 with 1x PBS.
    7. IR imaging and quantitation were performed using a LI-COR® Odyssey instrument using both 700 (SDH-A) and 800 (COX-1) according to the manufacturer’s instructions (Figure 2). Export data for analysis.
  6. The data were analyzed using GraphPad Prism software:
    1. Background-corrected resulted were obtained by subtracting the average background value which was calculated using all of the outer-wells that contained no compound treatment or antibodies as stated in the previous steps.
    2. 0.5% DMSO treated cells were used as a 100% (untreated) control.
    3. Individual protein levels were expressed as a percentage of the 0.5% DMSO control.
    4. In cases where cell viability was severely affected, the data for mitochondrial biogenesis were excluded from the analysis due to significant errors associated with low signals.
    5. Chloramphenicol was used as a positive control for the assay and a representative dose-response is shown in Figure 3.


      Figure 1. A representative 96-well plate map


      Figure 2. A representative 96-well plate IR image. The photo was adapted from the MitoSciences website on Jan 2012.


      Figure 3. Inhibition of mitochondrial biogenesis by chloramphenicol in PC-3 cells after 5-day culture. The % levels of COX-1 and SDH-A were shown in (•) and (▼), respectively. The cell viability, shown in (■), was monitored in parallel using another 96-well plate of PC-3 cells using CellTiter Glo. The data plotted were averages ± standard error from duplicate measurements. The CC50 values were 2.4, 13.4, and 88 µM for COX-1, ATP, and SDH-A levels, respectively.

Notes

  1. The Mitobiogenesis CC50 value was defined as the concentration at which individual protein level (COX-1 or SDH-A) decreased by 50% in comparison to the DMSO control. The data were analyzed using GraphPad Prism 5.0. CC50 values were calculated by non-linear regression analysis using a sigmoidal dose-response (variable slope) equation [four parameter logistic equation]: Y = Bottom + (Top-Bottom)/(1+10^((LogCC50-X)*HillSlope)] where the Bottom and Top values were fixed at 0 and 100, respectively. The Mitobiogenesis CC50 values were calculated as an average of two to three independent experiments.

Recipes

  1. Kaighn’s F12 medium
    Kaighn’s F12
    10% FBS
    100 units/ml penicillin and 100 µg/ml streptomycin (P/S)

Acknowledgments

All of the work was sponsored by Gilead Sciences, Inc. This protocol was adapted from Feng et al. (2014).

References

  1. Feng, J. Y., Cheng, G., Perry, J., Barauskas, O., Xu, Y., Fenaux, M., Eng, S., Tirunagari, N., Peng, B., Yu, M., Tian, Y., Lee, Y. J., Stepan, G., Lagpacan, L. L., Jin, D., Hung, M., Ku, K. S., Han, B., Kitrinos, K., Perron, M., Birkus, G., Wong, K. A., Zhong, W., Kim, C. U., Carey, A., Cho, A. and Ray, A. S. (2014). Inhibition of hepatitis C virus replication by GS-6620, a potent C-nucleoside monophosphate prodrug. Antimicrob Agents Chemother 58(4): 1930-1942.
  2. Instruction Manual of MitoToxTMMitoBiogenesisTM In-Cell Elisa Kit (IR) (Abcam, catalog number: ab110216). http://www.abcam.com/mitobiogenesisTM-in-cell-elisa-kit-ir-ab110216.html.

简介

药物诱导的线粒体损伤可以由许多不同的机制引起,包括线粒体DNA复制,转录,翻译和改变的蛋白质功能的抑制。 相对于细胞蛋白质合成的线粒体蛋白质合成水平或线粒体生物发生的确定提供了关于潜在线粒体毒性的重要信息。

关键字:线粒体毒性, 线粒体蛋白质合成, 核苷类药物相关毒性, 氯霉素, mitobiogenesis™细胞ELISA Kit

材料和试剂

  1. PC-3细胞(ATCC,CRL-1435,目录号:7348669)
  2. 化合物的三倍系列稀释物
  3. MitoTox TM MitoBiogenesis TM In-Cell ELISA Kit(Abcam,目录号:ab110216),包括
    1. 1,000x IRDye 标记的二抗(物种未被供应商披露)
    2. 200x一抗(物种未被供应商披露)
    3. 100x Triton X-100
    4. 10x阻塞缓冲区
    5. 10x磷酸盐缓冲盐水(PBS)
    6. 400x Tween-20
    7. 1x Janus绿色染色剂(可选)
  4. DMSO(细胞培养级)(Sigma-Aldrich,目录号:D2650)
  5. CellTiter Glo(Promega Corporation,目录号:G7571)
  6. Kaighn's F12(Life Technologies,Gibco ,目录号:21127)
  7. FBS(HyClone,目录号:SH30071.03)
  8. 100单位/ml青霉素和100μg/ml链霉素(P/S)(Life Technologies,Gibco ,目录号:15140-122)
  9. 细胞固定试剂:4%多聚甲醛(20%多聚甲醛,VWR International,目录号:15713-S)
  10. 氯霉素(10mM的DMSO溶液)(Sigma-Aldrich,目录号:C1919-5G)
  11. Kaighn's F12培养基(见配方)
    注意:将细胞维持在Kaighn's F12培养基中。 细胞每周传代两次并维持在90%汇合。 第4和第15代之间的细胞用于测定。

设备

  1. 96孔板(胶原I包被的96孔板)(VWR International,目录号:73521-028)
  2. 滴定板振荡器(Lab-line仪器,型号:4626)
  3. LI-COR ® Odyssey仪器(LI-COR)

软件

  1. GraphPad Prism 5.0(GraphPad软件)

程序

  1. 将PC-3细胞以2.5×10 3个细胞/孔的密度接种在处理前一天的胶原包被的96孔板中的每孔100μl的最终测定体积中。 />
  2. 在治疗当天,在接近化合物的一般细胞毒性5天CC 50值的浓度开始,在96孔板中一式两份制备化合物在DMSO中的三倍系列稀释液。 通过监测细胞ATP水平的变化来测量CC 50值。 对于具有一般细胞毒性CC 50值≥100μM的化合物,起始浓度为100μM。 代表性的96孔板图如图1所示。
    1. 用0.5%DMSO处理图1中标记为空白的外孔,并用作将来数据分析的背景。
    2. 将0.5%DMSO处理的细胞(图1中标记为DMSO对照)用作非药物处理的对照。
  3. 将化合物与培养基混合以制备2×化合物 - 培养基溶液,然后向96孔细胞板中的相关孔中加入100μl/孔的2×化合物 - 培养基溶液。 孔的最终体积为200μl。 最终的DMSO含量为0.5%
  4. 孵育5天后,将细胞用50μl/孔的4%多聚甲醛在室温下固定20分钟。 固定后,用1×PBS洗涤细胞两次(由稀释10×PBS制备)
    1. 此时,可以将板用100μl/孔的1×PBS在4℃下储存至多一周。
  5. 使用定量免疫细胞化学来测量培养细胞中复合物II(SDH-A)和IV(COX-1)的蛋白水平的MitoBiogenesis TM sup/In-Cell ELISA试剂盒分析细胞, 以下步骤:
    1. 用100μl/孔的1x透化处理使细胞透化 缓冲液(用1×PBS稀释100×Triton X-100制备)30分钟 min,在室温下在轻度摇动下在滴定板振荡器上   恒定速度(在Lab-line仪器上设置2)。 速度相同 当使用振动器时,在所有以下步骤中使用设置
    2. 从细胞板除去透化缓冲液后, 细胞与2x封闭缓冲液(200μl/孔)在37℃温育2小时 室温,在滴定板振荡器上轻轻搅拌
      1. 2×封闭缓冲液由1×PBS的10×封闭缓冲液的稀释制备。
    3. 除去封闭缓冲液后,将细胞与1x孵育   一抗在1x封闭缓冲液(100μl/孔)中4℃过夜 ℃,在滴定板振荡器上轻轻搅拌
      1. 1x封闭缓冲液通过用1x PBS稀释10x封闭缓冲液制备
      2. 1x一抗是用1x封闭缓冲液稀释200x一抗制备的。
      3. 用不含抗体的1x封闭缓冲液处理外孔。
    4. 第二天,用1×PBS-Tween缓冲液(300μl/孔,3次)洗涤细胞三次。
      1. 从用1x PBS稀释400x吐温-20制备1×PBS-Tween缓冲液。
    5. 洗涤后,将细胞与1×标记的IRDye温育 二抗在1x封闭缓冲液(100μl/孔)中室温2小时 在滴定板振荡器上轻轻搅拌温度
      1. 1x封闭缓冲液通过用1x PBS稀释10x封闭缓冲液制备
      2. 从稀释制备1×标记的次级抗体 的1000倍IRDye 标记的二抗与1x封闭缓冲液
      3. 用不含抗体的1x封闭缓冲液处理外孔。
    6. 用1×PBS-Tween缓冲液(300μl/孔)洗涤细胞5 并保持在1×PBS-Tween缓冲液(200μl/孔)中直到成像
      1. 从用1x PBS稀释400x吐温-20制备1×PBS-Tween缓冲液。
    7. 使用LI-COR Odyssey进行IR成像和定量 仪器使用700(SDH-A)和800(COX-1)根据 制造商的说明(图2)。 导出数据进行分析。
  6. 使用GraphPad Prism软件分析数据:
    1. 通过减去平均值获得背景校正的结果 背景值,其使用所有外井计算 不含如前所述的化合物处理或抗体 步骤。
    2. 将0.5%DMSO处理的细胞用作100%(未处理的)对照
    3. 单个蛋白质水平表示为0.5%DMSO对照的百分比。
    4. 在细胞活力受到严重影响的情况下, 线粒体生物发生被排除在分析之外 与低信号相关的显着误差。
    5. 氯霉素用作测定的阳性对照,代表性的剂量 - 反应如图3所示

      图1.代表性96孔板图


      图2.一个代表性的96孔板红外图像。照片摘自2012年1月的MitoSciences网站。


      图3.氯霉素对线粒体生物发生的抑制 5天培养后的PC-3细胞。 COX-1和SDH-A的%水平 分别显示在(•)和(▼)中。细胞活力,如(■)所示, 使用另一个96孔PC-3细胞板并行监测 使用CellTiter Glo。 绘制的数据为平均值±标准误差 来自重复测量。 CC 50值为2.4,13.4和88μM 分别为COX-1,ATP和SDH-A水平。

笔记

  1. 将Mitobiogenesis CC 50值定义为与DMSO对照相比,个体蛋白水平(COX-1或SDH-A)降低50%的浓度。 使用GraphPad Prism 5.0分析数据。 通过使用S形剂量 - 反应(可变斜率)方程[四参数对数方程]的非线性回归分析计算CC 50值:Y =底部+(顶部 - 底部)/(1 + 10 其中Bottom和Top值分别固定为0和100,计算有丝分裂生成CC 50值作为(对数的) 平均2〜3次独立实验。

食谱

  1. 凯恩的F12培养基
    Kaighn的F12
    10%FBS
    100单位/ml青霉素和100μg/ml链霉素(P/S)

致谢

所有的工作是由吉利德科学公司赞助的。该协议改编自Feng等人(2014)。

参考文献

  1. Feng,JY,Cheng,G.,Perry,J.,Barauskas,O.,Xu,Y.,Fenaux,M.,Eng,S.,Tirunagari,N.,Peng,B.,Yu,M.,Tian Y.,Lee,YJ,Stepan,G.,Lagpacan,LL,Jin,D.,Hung,M.,Ku,KS,Han,B.,Kitrinos,K.,Perron,M.,Birkus, ,Wong,KA,Zhong,W.,Kim,CU,Carey,A.,Cho,A.and Ray,AS(2014)。 通过GS-6620(一种有效的C-核苷单磷酸酯前药)抑制丙型肝炎病毒复制。 a> Antimicrob Agents Chemother 58(4):1930-1942。
  2. MitoTox TM MitoBiogenesis TM细胞内ELISA试剂盒(IR)(Abcam,目录号:ab110216)的说明书。 http://www.abcam .com/mitobiogenesis TM -in-cell-elisa-kit-ir-ab110216.html。
  • 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。
引用:Xu, Y. and Feng, J. Y. (2015). Mitochondrial Biogenesis Assay after 5-day Treatment in PC-3 Cells. Bio-protocol 5(2): e1378. DOI: 10.21769/BioProtoc.1378.
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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。