使用高含量显微镜的细胞氧化还原分析
1Laboratory of Cell Biology and Histology, Department of Veterinary Sciences,University of Antwerp, 2Cell Systems and Imaging Research Group (CSI), Department of Molecular Biotechnology,Ghent University, 3Department of Biochemistry , Radboud Institute for Molecular Life Sciences,Radboud University Medical Center
Summary
本文提出了一种高含量显微镜工作流程，用于同时定量细胞内ROS水平，以及线粒体膜电位和形态 – 共同称为线粒体形态功能 – 使用细胞透过性荧光报告分子5-（和 – 6） – 氯甲基-2'，7'-二氯二氢荧光素二乙酸酯，乙酰酯（CM-H 2 DCFDA）和四甲基罗丹明甲酯（TMRM）。
Abstract
活性氧（ROS）调节基本细胞过程，包括基因表达，迁移，分化和增殖。然而，过量的ROS水平诱导氧化应激状态，伴随着对DNA，脂质和蛋白质的不可逆的氧化损伤。因此，ROS的定量为细胞健康状况提供了直接依据。由于线粒体是ROS的主要细胞来源和靶标，所以在同一细胞中线粒体功能和ROS产生的联合分析对于更好地了解病理生理条件的互连至关重要。因此，开发了基于高含量显微镜的策略，用于同时定量细胞内ROS水平，线粒体膜电位（ΔΨm）和线粒体形态。它是基于自动化的宽场荧光显微镜和生物贴壁细胞的图像分析，生长在多孔板和staine中d与细胞可渗透的荧光报告分子CM-H 2 DCFDA（ROS）和TMRM（ΔΨm和线粒体形态）。与荧光测定法或流式细胞术相反，该策略允许在实验刺激前后具有高时空分辨率的个体细胞水平的亚细胞参数的定量。重要的是，该方法的基于图像的性质允许除了信号强度之外还提取形态学参数。组合特征集用于探索和统计多变量数据分析，以检测亚群，细胞类型和/或治疗之间的差异。在此，提供了测定的详细描述，以及证明其在化学扰动之后在细胞状态之间明确区分的潜力的示例性实验。
Introduction
通过ROS产生和ROS消除系统之间的动态相互作用，精细调节细胞内ROS的浓度。两者之间的不平衡引起氧化应激状态。线粒体的主要来源之一是线粒体1 。鉴于它们在细胞呼吸中的作用，它们负责大量的细胞内超氧化物（O 2 · – ）分子2 。这主要是由于电子传输链的复合物1在强的负内线粒体膜电位（Δψm）， 即线粒体超极化的条件下，电子泄漏到O 2 。另一方面，线粒体去极化也与增加的ROS生产相关，指向多种作用模式3,4,5 ，> 6，7，8 。此外，通过裂解融合机理的蛋白质的氧化还原修饰，ROS共调节线粒体形态9。例如，碎片与增加的ROS产生和凋亡相关， 10,11 ，而丝状线粒体已经与营养物质饥饿和保护相关mitophagy 12 。鉴于细胞ROS和线粒体形态功能之间的复杂关系，两者应理想地在活细胞中同时量化。为了做到这一点，基于自动化宽视野显微镜和用荧光探针CM-H 2 DCFDA（ROS）和TMRM（线粒体Δψm和形态）染色的贴壁细胞培养物的图像分析开发了高含量成像测定。高含量成像是指提取sp关于使用多个互补标记和自动图像分析的细胞表型的信息丰富（ 即大量描述性特征）。当与自动显微镜组合时，可以并行筛选许多样品（ 即高通量），从而增加测定的统计学功能。实际上，协议的主要资产是它允许在同一个信元中同时量化多个参数，而这对于大量的信元和条件来说是可以的。
协议分为8个部分（以下协议详细描述）：1）96孔板中的播种细胞; 2）储备溶液，工作溶液和成像缓冲液的制备; 3）设置显微镜; 4）用CM-H 2 DCFDA和TMRM装载细胞; 5）首次实时成像测量基础ROS水平和线粒体形态功能; 6）加入叔丁基后进行第二次活体成像过氧化物（TBHP）测量诱导的ROS水平; 7）自动图像分析; 8）数据分析，质量控制和可视化。
该测定法最初是针对正常人皮肤成纤维细胞（NHDF）开发的。由于这些细胞是大而平坦的，它们非常适合于评估2D宽幅图像13,14 中的线粒体形态。然而，通过轻微修改，该方法适用于其他贴壁细胞类型。此外，在CM-H 2 DCFDA和TMRM的组合之后，工作流程符合各种具有不同分子特异性的荧光染料对1,15。
Protocol
对于NHDF细胞和使用材料文件中指定的多孔板，描述了下面的方案。有关工作流程的总体概述，请参见图1 。 1.试剂的制备通过用10％v / v胎牛血清（FBS）和100IU / mL青霉素和100IU / mL链霉素（PS）补充Dulbecco's Modified Eagle Medium（DMEM）来制备完整培养基。对于500 mL完全培养基，加入50mL FBS和5 mL PS至445 mL DMEM。 通过用20mM HEPES补充汉克平衡盐溶液…
Representative Results
该测定已经使用几个对照实验进行了基准测试，其结果在Sieprath et al。 1 。简而言之，CM-H 2 DCFDA和TMRM的荧光响应分别定量分析了细胞内ROS和Δψm的变化，以确定其动态范围。对于CM-H 2 DCFDA，当用10μM至160μM范围内的增加的TBHP浓度处理时，NHDF显示出荧光信号的线性增加。同样，对于TMRM，当用1〜10μg/μL范围内增加浓度的寡霉素（其诱?…
Discussion
本文介绍了用于同时定量NHDF中细胞内ROS水平和线粒体形态功能的高含量显微镜方法。通过对SQV处理的NHDF的案例研究证明其性能。结果支持早期的文献证据，其中在用1型HIV蛋白酶抑制剂治疗后已经观察到ROS水平升高或线粒体功能障碍，尽管在单独的实验19,20,21,22,23,24,25中。重要的区别是所描述的测定能够在相同的活细胞中同时测量这些参数，以及形态学数据。这种方法的主要优点在于它们在空间?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by the University of Antwerp (TTBOF/29267, TTBOF/30112), the Special Research Fund of Ghent University (project BOF/11267/09), NB-Photonics (Project code 01-MR0110) and the CSBR (Centers for Systems Biology Research) initiative from the Netherlands Organization for Scientific Research (NWO; No: CSBR09/013V). Parts of this manuscript have been adapted from another publication1, with permission of Springer. The authors thank Geert Meesen for his help with the widefield microscope.
Materials
| Reagents | |||
| Tetramethylrhodamine, Methyl Ester, Perchlorate (TMRM) | ThermoFisher Scientific | T668 | |
| CM-H2DCFDA (General Oxidative Stress Indicator) | ThermoFisher Scientific | C6827 | |
| Dimethyl sulfoxide | Sigma)Aldrich | D8418 | |
| MatriPlate 96-Well Glass Bottom MicroWell Plate 630 µL-Black 0.17 mm Low Glass Lidded | Brooks life science systems | MGB096-1-2-LG-L | |
| HBSS w/o Phenol Red 500 ml | Lonza | BE10-527F | |
| DMEM high glucose with L-glutamine | Lonza | BE12-604F | |
| Phosphate Bufered Saline (PBS) w/o Ca and Mg | Lonza | BE17-516F | |
| HEPES 1M 500mL | Lonza | 17-737F | |
| Trypsin-Versene (EDTA) Solution | Lonza | BE17-161E | |
| Cy3 AffiniPure F(ab')₂ Fragment Donkey Anti-Rabbit IgG (H+L) | Jackson | 711-166-152 | Antibody used for acquiring flat-field image |
| Alexa Fluor 488 AffiniPure F(ab')₂ Fragment Donkey Anti-Rabbit IgG (H+L) | Jackson | 711-546-152 | Antibody used for acquiring flat-field image |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Nikon Ti eclipse widefield microscope | Nikon | ||
| Perfect Focus System (PFS) | Nikon | hardware-based autofocus system | |
| CFI Plan Apo Lambda 20x objective | Nikon | ||
| Name | Company | Catalog Number | Comments |
| Software | |||
| NIS Elelements Advanced Research 4.5 with JOBS module | Nikon | This software is used to steer the microscope and program/perform the automatic image acquisition prototocol | |
| ImageJ (FIJI) Version 2.0.0-rc-43/1.50g | |||
| RStudio Version 1.0.44 | Rstudio | ||
| R version 3.3.2 |
References
- Sieprath, T., Corne, T. D. J., Willems, P. H. G. M., Koopman, W. J. H., De Vos, W. H. Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction. AAEC. 219, 149-177 (2016).
- Marchi, S., et al. Mitochondria-ros crosstalk in the control of cell death and aging). J Signal Transduct. 2012, 1-17 (2012).
- Korshunov, S. S., Skulachev, V. P., Starkov, A. A. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS lett. 416 (1), 15-18 (1997).
- Miwa, S., Brand, M. D. Mitochondrial matrix reactive oxygen species production is very sensitive to mild uncoupling. Biochem Soc Trans. 31 (Pt 6), 1300-1301 (2003).
- Verkaart, S., et al. Superoxide production is inversely related to complex I activity in inherited complex I deficiency. BBA-GEN SUBJECTS. 1772 (3), 373-381 (2007).
- Murphy, M. P. How mitochondria produce reactive oxygen species. Biochem J. 417 (pt 1), 1-13 (2009).
- Lebiedzinska, M., et al. Oxidative stress-dependent p66Shc phosphorylation in skin fibroblasts of children with mitochondrial disorders. BBA-GEN SUBJECTS. 1797 (6-7), 952-960 (2010).
- Forkink, M., et al. Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II, III, IV and V. BBA-BIOENERGETICS. 1837 (8), 1247-1256 (2014).
- Willems, P. H. G. M., Rossignol, R., Dieteren, C. E. J., Murphy, M. P., Koopman, W. J. H. Redox Homeostasis and Mitochondrial Dynamics. Cell Metab. 22 (2), 207-218 (2015).
- Koopman, W. J. H., et al. Human NADH:ubiquinone oxidoreductase deficiency: radical changes in mitochondrial morphology?. Am J Physiol Cell Physiol. 293 (1), C22-C29 (2007).
- Archer, S. L. Mitochondrial dynamics–mitochondrial fission and fusion in human diseases. N Engl J Med. 369 (23), 2236-2251 (2013).
- Rambold, A. S., Kostelecky, B., Elia, N., Lippincott-Schwartz, J. Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. Proc Natl Acad Sci U S A. 108 (25), 10190-10195 (2011).
- Koopman, W. J. H., et al. Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2 ",7 -"dichlorofluorescein. Cytometry A. 69A (12), 1184-1192 (2006).
- Iannetti, E. F., Smeitink, J. A. M., Beyrath, J., Willems, P. H. G. M., Koopman, W. J. H. Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy. Nat Protoc. 11 (9), 1693-1710 (2016).
- Sieprath, T., et al. Sustained accumulation of prelamin A and depletion of lamin A/C both cause oxidative stress and mitochondrial dysfunction but induce different cell fates. Nucleus. 6 (3), 236-246 (2015).
- Zuiderveld, K. Contrast limited adaptive histogram equalization. Graphics gems IV. , 474-485 (1994).
- Chang, W., Cheng, J., Allaire, J. J., Xie, Y., McPherson, J. . shiny: Web Application Framework for R. R package version 0.14.2. , (2017).
- Konietschke, F., Placzek, M., Schaarschmidt, F., Hothorn, L. A. nparcomp: An R Software Package for Nonparametric Multiple Comparisons and Simultaneous Confidence Intervals. J Stat Softw. 64 (9), 1-17 (2015).
- Estaquier, J., et al. Effects of antiretroviral drugs on human immunodeficiency virus type 1-induced CD4(+) T-cell death. J Virol. 76 (12), 5966-5973 (2002).
- Matarrese, P., et al. Mitochondrial membrane hyperpolarization hijacks activated T lymphocytes toward the apoptotic-prone phenotype: homeostatic mechanisms of HIV protease inhibitors. J Immunol. 170 (12), 6006-6015 (2003).
- Roumier, T., et al. HIV-1 protease inhibitors and cytomegalovirus vMIA induce mitochondrial fragmentation without triggering apoptosis. Cell Death Differ. 13 (2), 348-351 (2006).
- Chandra, S., Mondal, D., Agrawal, K. C. HIV-1 protease inhibitor induced oxidative stress suppresses glucose stimulated insulin release: protection with thymoquinone. Exp Biol Med (Maywood). 234 (4), 442-453 (2009).
- Touzet, O., Philips, A. Resveratrol protects against protease inhibitor-induced reactive oxygen species production, reticulum stress and lipid raft perturbation. AIDS. 24 (10), 1437-1447 (2010).
- Bociąga-Jasik, M., et al. Metabolic effects of the HIV protease inhibitor–saquinavir in differentiating human preadipocytes. Pharmacol Rep. 65 (4), 937-950 (2013).
- Xiang, T., Du, L., Pham, P., Zhu, B., Jiang, S. Nelfinavir, an HIV protease inhibitor, induces apoptosis and cell cycle arrest in human cervical cancer cells via the ROS-dependent mitochondrial pathway. Cancer Lett. 364 (1), 79-88 (2015).
- Blanchet, L., Smeitink, J. A. M., et al. Quantifying small molecule phenotypic effects using mitochondrial morpho-functional fingerprinting and machine learning. Sci. Rep. 5, 8035 (2015).
- . Reactive Oxygen Species (ROS) Detection Reagents Available from: https://tools.lifetechnologies.com/content/sfs/manuals/mp36103.pdf (2006)
- Koh, C. M. Preparation of cells for microscopy using cytospin. Methods Enzymol. 533, 235-240 (2013).
- Mihara, K., Nakayama, T., Saitoh, H. A Convenient Technique to Fix Suspension Cells on a Coverslip for Microscopy. Curr Protoc Cell Biol. 68, 4.30.1-4.30.10 (2015).
- Deutsch, M., Deutsch, A., et al. A novel miniature cell retainer for correlative high-content analysis of individual untethered non-adherent cells. Lab Chip. 6 (8), 995-996 (2006).
- Price, H. P., MacLean, L., Marrison, J., O’Toole, P. J., Smith, D. F. Validation of a new method for immobilising kinetoplastid parasites for live cell imaging. Mol Biochem Parasitol. 169 (1), 66-69 (2010).
- Sabati, T., Galmidi, B. S., Korngreen, A., Zurgil, N., Deutsch, M. Real-time monitoring of changes in plasma membrane potential via imaging of fluorescence resonance energy transfer at individual cell resolution in suspension. JBO. 18 (12), (2013).
- Fercher, A., O’Riordan, T. C., Zhdanov, A. V., Dmitriev, R. I., Papkovsky, D. B. Imaging of cellular oxygen and analysis of metabolic responses of mammalian cells. Meth Mol Biol. 591 (Chapter 16), 257-273 (2010).
- Graf, R., Rietdorf, J., Zimmermann, T. Live cell spinning disk microscopy. Adv. Biochem. Eng. Biotechnol. 95 (Chapter 3), 57-75 (2005).
- Gao, L., Shao, L., Chen, B. C., Betzig, E. 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat. Protoc. 9 (5), 1083-1101 (2014).
- Meijer, M., Hendriks, H. S., Heusinkveld, H. J., Langeveld, W. T., Westerink, R. H. S. Comparison of plate reader-based methods with fluorescence microscopy for measurements of intracellular calcium levels for the assessment of in vitro neurotoxicity. Neurotoxicology. 45, 31-37 (2014).
- Bushway, P. J., Mercola, M., Price, J. H. A comparative analysis of standard microtiter plate reading versus imaging in cellular assays. Assay and drug development technologies. 6 (4), 557-567 (2008).
- Black, C. B., Duensing, T. D., Trinkle, L. S., Dunlay, R. T. Cell-Based Screening Using High-Throughput Flow Cytometry. Assay Drug Dev Technol. 9 (1), 13-20 (2011).
Cite This Article
Sieprath, T., Corne, T., Robijns, J., Koopman, W. J. H., De Vos, W. H. Cellular Redox Profiling Using High-content Microscopy. J. Vis. Exp. (123), e55449, doi:10.3791/55449 (2017).