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Measuring Mitochondrial ROS in Mammalian Cells with a Genetically Encoded Protein Sensor
用基因编码的蛋白质传感器测定哺乳动物细胞线粒体中的ROS   

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EMBO Molecular Medicine
May 2016

Abstract

Reactive oxygen species (ROS) are not only known for their toxic effects on cells, but they also play an important role as second messengers. As such, they control a variety of cellular functions such as proliferation, metabolism, differentiation and apoptosis. Thus, ROS are involved in the regulation of multiple physiological and pathophysiological processes. It is now apparent that there are transient and local changes in ROS in the cell; in so-called ‘microdomains’ or in specific cellular compartments, which affect signaling events. These ROS hotspots need to be studied in more depth to understand their function and regulation. Therefore, it is necessary to identify and quantify redox signals in single cells with high spatial and temporal resolution. Genetically encoded fluorescence-based protein sensors provide such necessary tools to examine redox-signaling processes. A big advantage of these sensors is the possibility to target them specifically. Mitochondria are essential for energy metabolism and are one of the major sources of ROS in mammalian cells. Therefore, the evaluation of redox potential and ROS production in these organelles is of great interest. Herein, we provide a protocol for the real-time visualization of mitochondrial hydrogen peroxide (H2O2) using the H2O2-specific ratiometric sensor mitoHyPer in adherent mammalian cells.

Keywords: Mitochondrial ROS (线粒体ROS), Protein sensor (蛋白传感器), Fluorescence microscopy (显微镜检查), Real-time imaging (实时成像), Mammalian cell (哺乳动物细胞), HyPer (HyPer), roGFP-Orp1 (roGFP-Orp1)

Background

ROS are produced as by-products of mitochondrial respiration, through the leakage of electrons from the electron transfer chain. These ROS are considered toxic and cause the oxidation of lipids, proteins, and lead to mitochondrial DNA damage (Ralph et al., 2010; Bogeski and Niemeyer, 2014; Cierlitza et al., 2015; Gibhardt et al., 2016). While mitochondria serve as a hub of metabolism, bioenergetics, and cell death, the emerging role of mitochondrial ROS as second messengers in regulating other cellular functions is also increasingly accepted (Chandel, 2015; Reczek and Chandel, 2015; Shadel and Horvath, 2015; Wilems et al., 2015). To monitor mitochondrial ROS with high spatial and temporal resolution remains challenging due to the short half-life of ROS and the limitation of available probes (Kuznetsov et al., 2011; Norcross et al., 2017). The primary reactive species of mitochondrial origin are superoxide anion, hydroxyl radical, singlet oxygen, and hydrogen peroxide (Gibhardt et al., 2016; Idelchik et al., 2017). Hydrogen peroxide (H2O2) is one of the most stable ROS and is thus an attractive tracking tool for examining the cellular redox state.

During the past decade, several groups designed genetically encoded protein sensors to specifically detect H2O2 (Belousov et al., 2006; Gutscher et al., 2009). The specificity, reversibility, and sensitivity of these protein sensors make them suitable for real-time visualization of H2O2 under a broad range of physiological conditions and stimulations.

The HyPer and roGFP2-Orp1 sensors are advantageous in particular and can be used in various cell systems (Ermakova et al., 2014; Hernandez-Barrera et al., 2013; Bogeski et al., 2016). The HyPer sensor is a combination of a circular permutated yellow fluorescent protein (cpYFP), which is inserted in the regulatory domain of the bacterial H2O2 sensing protein OxyR. The oxidation of cysteine199 found on OxyR initiates conformational changes in HyPer. In a reduced state HyPer has two excitation peaks at 420 nm and 500 nm, and one emission peak at 516 nm. Following oxidation, the peak at 420 nm decreases and the peak at 500 nm increases, thus allowing ratiometric measurement of H2O2. (Bilan and Belousov, 2017). Given that pH fluctuations can also affect the signal from HyPer probes, a mutation at cysteine 199 was introduced to generate a probe named SypHer for monitoring pH, which has the same pH sensitivity but does not react to oxidation (Matlashov et al., 2015; Poburko et al., 2011). The roGFP probe is based on an engineered GFP containing two cysteine residues capable of forming a disulfide bond (Morgan et al., 2011). It has two excitation maxima at 400 and 490 nm with the emission around 510 nm; the ratio of these two excitation maxima depends on the state of the disulfide bond. The development of roGFP probes now provides important alternative tools aimed at detecting H2O2 or the potential of the glutathione redox pair (Gutscher et al., 2008; Kasozi et al., 2013; Habich and Riemer, 2017; Lismont et al., 2017; Müller et al., 2017).

Here we describe a detailed protocol for the real-time imaging and monitoring of mitochondrial H2O2 with the mitoHyPer sensor. The approach can be performed on different cellular systems with a basic understanding of real-time imaging and fluorescence microscopy; the data analysis procedure depends on the software available.

Materials and Reagents

  1. Round glass coverslips 25 mm No. 1.5 (Kindler/ORSA tec®, Round cover glasses)
  2. 6-well plates (Corning, Costar®, catalog number: 3516 )
  3. Falcon tubes (15 ml) (VWR, Corning, catalog number: 62406-200 )
  4. Serological pipettes (Corning, Costar®, catalog number: 4488 )
  5. Plasmids
    mitoHyPer (Evrogen, catalog number: FP942 )
    mitoSypHer (Addgene, catalog number: 48251 )
  6. Cell growth medium (specific to the cells used in the experiment)
  7. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  8. Fugene® HD (Promega, catalog number: E2312 )
  9. Opti-MEMTM (Thermo Fisher Scientific, GibcoTM, catalog number: 51985-026 )
  10. Baysilone paste (VWR, GE Bayer Silicines, catalog number: 291-1210 )
  11. Accutase (Sigma-Aldrich, catalog number: A6964 ) or Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 25300062 )
  12. 1x DPBS, no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-094 )
  13. 1,4-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  14. Hydrogen peroxide solution 30% (w/w) in H2O, contains stabilizer (Sigma-Aldrich, catalog number: H1009 )
  15. Stimulants and inhibitors (these are experiment-dependent)
  16. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  17. Potassium chloride (KCl) (VWR, AnalaR NORMAPUR®, catalog number: 26764.298 )
  18. Calcium chloride dihydrate (CaCl2·2H2O) (Merck, catalog number: 102382 )
  19. Magnesium chloride (MgCl2) (Merck, catalog number: 105833025 )
  20. D(+)-Glucose anhydrous (Merck, catalog number: 108337 )
  21. EGTA (Sigma-Aldrich, catalog number: E4378 )
  22. 1 M HEPES (Sigma-Aldrich, catalog number: H7523 )
  23. Ringer buffer (0.25 mM Ca2+, pH 7.4) (see Recipes)

Equipment

  1. ZTM Series COULTER COUNTER® Cell (Beckman Coulter, model: 6605699 ) and Particle Counter Z1 (Beckman Coulter or any other counting device)
  2. Incubator with humidity and gas control for cell culture
  3. Zeiss Axio Observer.Z1 (Carl Zeiss, model: Axio Observer.Z1 ) setup (Figure 1) (Incubation System S includes Temp Module S, CO2 Module S, O2 Module S, Heating Module S)
  4. Tweezers (e.g., style Dumont Nr. 7)
  5. Imaging chamber and ring insert (self-made) and perfusion system (Figure 2)


    Figure 1. Zeiss Cell Observer.Z1 setup with temperature, CO2 controlling unit, gas chamber and perfusion system. A. Analysis computer; B. Cell Observer.Z1 with 40x oil objective and corresponding filter sets; C. Evolve 512 x 512 EM-CCD camera; D. CO2 supply unit; E. Pecon XL S1 incubator and control modules; F. LED Colibri with corresponding modules. G. Pump and perfusion system.
    Notes:
    1.  For HyPer measurements, the CFP/YFP filters are essential, but a multiband filter cube with the same property is also a functional option.
    2. For HyPer experiments, we used the LED light source with the wavelength at 505 nm and 420 nm and corresponding beam splitters.


      Figure 2. Imaging chamber and mount module with temperature control. A. The self-made imaging chamber (i) with a perfusion chamber plastic insert (ii) that is fixed with knobs (iii). The coverslip with the cells is attached to the lower part of the plastic insert and a small 12 mm coverslip is attached to the upper part of the plastic insert in order to create a small perfusion channel for the measurement. B. The imaging chamber attached to the perfusion system and to the stage of the microscope (i). The perfusion tube (ii) is attached to a syringe in order to add the solutions during the measurement, while the second perfusion tube (iii) is attached to a suction pump system to remove the waste liquid.

Software

  1. Axiovision 4.6v (Zeiss) with a license for fast acquisition function and measurement analysis or similar

Procedure

  1. Day 1: Cell culture and seeding
    This protocol is exemplary for adherent cells, which can be transfected with reagents such as Fugene® HD. For cell lines which are difficult to transfect, we recommend an alternative transfection method (e.g., nucleofection by electroporation). If stable cells expressing the desired sensors are available, they can also be used for the imaging experiment as described in this protocol.
    1. Culture the cells with their corresponding growth medium until they reach a confluency of around 70%. Remove the growth medium, wash the cells once with 5 ml DPBS and detach the cells by incubating them with 1 ml trypsin or 1 ml accutase (as used for their normal cultivation) at room temperature.
    2. Suspend the cells in growth medium and dilute 100 µl of the suspension with DPBS to a ratio of 1:100 in a total volume of 10 ml. Determine the concentration of cells in the dilution with a Z1 cell counter or hemocytometer.
    3. Place autoclaved glass coverslips into a 6-well plate and seed 400,000 cells (in this example HEK293 cells) for each well in 2 ml of growth medium. Place the plate in a humidified cell culture incubator (37 °C, 5% CO2) and incubate overnight.

  2. Day 2: Transfection
    1. Remove the Opti-MEM medium and Fugene® HD solutions from the freezer and equilibrate both at room temperature for several minutes.
    2. Mix 100 µl of Opti-MEM medium with 4 to 10 µl of Fugene® HD solution (according to the manufacturer’s protocol), add the suggested amount of plasmid DNA (1 µg/µl endotoxin-free stock solution) to the mixture (1 µg/well is recommended, but the optimal amount can vary and depends on the cell type and plasmid). Pipette the mix up and down 15 times.
      Note: The optimal transfection conditions e.g., cell density, DNA amount, DNA:Fugene® HD ratio might need optimization for the cell line of choice.
    3. Wait for 15 min at room temperature, then add 100 µl of the transfection mixture to each well.
      Notes:
      1. If your cell growth medium contains antibiotics, it is advisable to change this before the transfection to growth medium without antibiotics, because they might reduce the transfection efficiency; otherwise, it is not necessary to change the growth medium before the transfection mixture is added.
      2. Since mitoHyPer and mitoSypHer have the same spectrum features, they should be transfected separately (in different wells).
    4. The cells are incubated in a humidified cell culture incubator (37 °C, 5% CO2). Change the medium in the transfected wells after 6 h with fresh cell growth medium. Keep the cells in the incubator until ready for imaging (37 °C, 5% CO2), for about 24-48 h.

  3. Day 3 or 4: Imaging
    Imaging is performed with a Zeiss Cell Observer.Z1 setup with temperature, CO2 controlling unit, gas chamber and perfusion system (Figure 1).
    1. Gently remove a cell-covered coverslip with a pair of delicate tweezers (avoiding the scrapping of cells in the central imaging area of the coverslip). Add Baysilone-paste on the edge of the bottom of the perfusion chamber plastic insert (self-made) and attach it to the coverslip (cells-facing-up). Fix a 12 mm coverslip with Baysilone-paste on the upper part of the plastic insert in order to create a small perfusion channel. Then fix the plastic insert (holding the coverslips) with the knobs and place the assembled chamber into the metal imaging chamber (see Figure 2A).
      Note: If simple experiments are performed (e.g., analyzing the resting levels with subsequent addition of saturating H2O2) it might be sufficient to use standard imaging chambers or glass-bottom plates and stimulate the signal changes by addition of the agents carefully with a pipette. However, for more experiments involving, multiple additions or washing-out experiments, a perfusion system is recommended.
    2. Place the imaging chamber on the microscope stage and attach the solution containing perfusion tubes (to avoid air in the system), on opposite sides of the chamber (as shown in Figure 2B). Perfuse gently with 2 ml Ringer solution (see Recipes) to wash away detached cells. Wait for 5 min to reach a CO2 (5 %) and temperature (37 °C) equilibrium before additional handling.
      Note: CO2 and temperature are controlled and monitored by the imaging system with the corresponding controlling units. Our perfusion system has on one side a syringe to apply the Ringer solution by hand and on the other side a suction pump to remove the waste.
    3. Using a 40x objective, search for a proper field of view that allows you to assess separate and well attached cells. Set the LED strength in order to get a proper signal, but not too high to avoid photobleaching of the sensor. Optimize the exposure time to obtain good image quality (signal over background) and keep the ratio of exposure time for both channels (420 nm vs. 505 nm) as a constant for all experiments. This part of the procedure will require some time to optimize, based on the cell types used and on the equipment available, since the light source and camera can vary.
    4. Start the experiment by measuring the resting level of H2O2 in the cells every 1 sec for at least 10 sec, then add stimulating substances through the perfusion system and record until the signal stabilizes (or according to the stimulation protocol). The frame number per minute and total imaging time should be optimized to achieve proper temporal resolution but also to avoid photobleaching.
      Note: The stimulating substances leading to the production of ROS from mitochondria vary depending on the scientific question and the cell type. For other scientific questions, only the resting redox level (e.g., the physiological H2O2 concentration under normal conditions) might be of interest.
    5. At the end of each measurement, a single dose of saturating H2O2 (e.g., 1 mM) should be added as a positive control and to determine the maximal intensity of the sensor (this might be needed for calibrating the system). To detect the fluorescence intensity of a fully reduced sensor (which will indicate if the sensor is already oxidized during resting conditions and provide the minimum value for calibration), we advise adding a reducing agent (e.g., 2 mM DTT) at the end of the experiment.
    6. Perform the same imaging procedure with the mitoSypHer sensor as an imaging control, since the HyPer sensor can be affected by changes in pH.
      Note: If the signal (or ratio change) obtained during the experiment with the HyPer sensor is due to oxidation, you will not see any changes in the SypHer signal during the same experimental conditions. If the pH influences your results, you will see also changes in the ratio of the SypHer sensor. Since HyPer is a pH-dependent sensor, this control is mandatory in order to discuss the data regarding redox changes.

Data analysis

Analysis with the Axiovision software

  1. Background correction
    The background correction should be performed by subtracting intensity values in a background ROI from a target (cell-based) ROI (Figure 3).


    Figure 3. Analysis example of HEK293 cells expressing mitoHyPer (also see Figure 4). A. Merged image (420 nm green, 505 nm red); B. exemplary presentation of analysis. The red circle represents the background ROI in a cell free region, while the borders of some cells are marked with white freehand drawing ROI for analysis.

  2. Ratiometric analysis
    The ratio kinetic curve is generated with the equation:



    using the Axiovision software.
    Note: The data analysis can be performed with different softwares from other suppliers. The basic calculation for the HyPer ratio can also be performed using an open source software such as ImageJ (https://imagej.nih.gov).
    The data are usually presented as mean ± SEM (or SD), and tested for significance with two-sided Student’s t-test. For each condition, at least three experiments should be performed with proper replicates.
  3. Below are representative images (A) with a magnification of a single cell (B), a graphed summary (C) and the statistical analysis (D) of what is expected from HEK293 cells following H2O2 and DTT addition (Figure 4).
    Note: If the probe is fully oxidized during the measurement and could not respond to saturating H2O2 at the end of the experiment, the result should be excluded from analysis. Since the pH is monitored with the SypHer probe, any experiment with significant fluctuations in pH should be excluded from analysis.


    Figure 4. Exemplary ROS measurement of HEK293 cells expressing the mitochondrial H2O2 sensor mitoHyPer. HEK293 cells were transfected with the mitoHyPer sensor using a Fugene® HD-based solution, 48 h prior to imaging. The cells were first titrated with 50 μM and 500 μM H2O2 for oxidation of the probe. Following the washout of H2O2, the cells were titrated with 2 mM DTT for the reduction of the probe. The change in fluorescence intensity ratio is represented as merged images (420 nm green, 505 nm red). As shown in (A), the addition of H2O2 caused oxidation of the probe and increased the signal ratio, while the addition of DTT reduced the signal ratio. The indicated region in (A) is shown magnified in (B). Since mitochondrial H2O2 is generated during a cell’s resting state, the probe can be partially oxidized by the constitutively generated ROS and can be reduced by membrane-permeant reducing agents such as DTT. The time course corresponding to the images in (A) and (B) is shown in (C) and the statistical analysis (mean ± SEM, n = 17) in (D). Scale bars = 10 µm.

Recipes

  1. Ringer buffer (0.25 mM Ca2+, pH 7.4)
    155 mM NaCl
    4.5 mM KCl
    10 mM glucose
    5 mM HEPES
    2.75 mM MgCl2
    0.25 mM CaCl2

Acknowledgements

This work was supported by the German Research Foundation (DFG) through SFB1190 project 17, SFB1027 project C4 and BO3643/3-2 research grant (all to IB). The authors declare no conflicts of interest or competing financial interests.

References

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  2. Bilan, D. S. and Belousov, V. V. (2017). New tools for redox biology: From imaging to manipulation. Free Radic Biol Med 109: 167-188.
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简介

活性氧(ROS)不仅以其对细胞的毒性作用而闻名,而且作为第二信使也起着重要的作用。如此,它们控制多种细胞功能,例如增殖,代谢,分化和凋亡。因此,ROS参与多种生理和病理生理过程的调节。现在很明显,细胞内存在ROS的短暂和局部变化;在所谓的“微域”或特定的细胞区室中,其影响信号传导事件。这些ROS热点需要更深入的研究,以了解其功能和规定。因此,有必要以高空间和时间分辨率在单个细胞中识别和量化氧化还原信号。遗传编码的基于荧光的蛋白质传感器提供了检测氧化还原信号传导过程的必要工具。这些传感器的一个很大的优势是可以针对他们。线粒体是能量代谢所必需的,并且是哺乳动物细胞中ROS的主要来源之一。因此,对这些细胞器中氧化还原电位和ROS产生的评估是非常有意义的。在此,我们提供了一个使用H 2 O 2特异性比例传感器mitoHyPer在贴壁哺乳动物细胞中。

【背景】ROS是通过线粒体呼吸的副产物,通过电子从电子传递链泄漏而产生的。这些ROS被认为是有毒的,并导致脂质,蛋白质的氧化,并导致线粒体DNA损伤(Ralph et al。,2010; Bogeski等人,2016; Bogeski ,2014; Monika et al。,2015)。虽然线粒体作为新陈代谢,生物能量学和细胞死亡的枢纽,线粒体ROS作为调节多种细胞功能的第二信使的新兴作用也日益被接受(Chandel,2015; Reczek和Chandel,2015; Shadel和Horvath,2015; Wilems 等人,2015)。由于ROS的半衰期短以及可用探针的限制(Kuznetsov等人,2011; Norcross等人,<! - SIPO
在过去的十年中,几个小组设计了遗传编码的蛋白质传感器来专门检测H 2 O 2(Belousov等人,2006; Gutscher,等人,2009)。这些蛋白质传感器的特异性,可逆性和灵敏度使得它们适合在广泛的生理条件和刺激下实时观察H 2 O 2 2。

HyPer和roGFP2-Orp1传感器是特别有利的,并且可以用于各种细胞系统(Ermakova等人,2014; Hernandez-Barrera等人,2013; Bogeski et al。,2016)。 HyPer传感器是一种圆形置换黄色荧光蛋白(cpYFP)的组合,其插入在细菌H 2 O 2感受蛋白OxyR的调节结构域中。在OxyR上发现的半胱氨酸199的氧化引发HyPer的构象变化。在还原态HyPer在420nm和500nm具有两个激发峰,在516nm具有一个发射峰。在氧化之后,在420nm处的峰减小并且在500nm处的峰增加,从而允许H 2 O 2:2的比率测量。 (Bilan和Belousov,2017)。考虑到pH波动也可以影响来自HyPer探针的信号,引入半胱氨酸199处的突变以产生名为SypHer的用于监测pH的探针,其具有相同的pH敏感性但是不与氧化反应(Matlashov等人, ,2015; Poburko et al。,2011)。 roGFP探针基于工程化的GFP,其含有能够形成二硫键的两个半胱氨酸残基(Morgan等人,2011)。它在400和490nm有两个激发最大值,发射在510nm附近;这两个激发极大值的比例取决于二硫键的状态。 roGFP探针的发展现在提供了用于检测H 2 O 2或谷胱甘肽氧化还原对的潜力的重要替代工具(Gutscher等人 >,2008; Kasozi等人,2013; Habich等人,2017; Lismont等人,2017;Müller等人, et al。,2017)。

在这里,我们描述了mitoHyPer传感器实时成像和监测线粒体H 2 O 2的详细方案。该方法可以在不同的细胞系统上进行实时成像和荧光显微镜的基本理解;数据分析程序取决于可用的软件。

关键字:线粒体ROS, 蛋白传感器, 显微镜检查, 实时成像, 哺乳动物细胞, HyPer, roGFP-Orp1

材料和试剂

  1. 圆形玻璃盖玻片25 mm 1.5号(Kindler / ORSA tec ®,圆形眼镜
  2. 6孔板(Corning,Costar ®,目录号:3516)
  3. 猎鹰管(15毫升)(VWR,康宁,目录号:62406-200)
  4. 血清学移液器(Corning,Costar ,目录号:4488)
  5. 质粒
    mitoHyPer(Evrogen,目录号:FP942)
    mitoSypHer(Addgene,目录号:48251)
  6. 细胞生长培养基(特异于实验中使用的细胞)
  7. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  8. Fugene HD(Promega,目录号:E2312)
  9. Opti-MEM TM(Thermo Fisher Scientific,Gibco TM,目录号:51985-026)。
  10. Baysilone粘贴(GE拜耳有机硅,超链接供应商
  11. Accutase(Sigma-Aldrich,目录号:A6964)或胰蛋白酶(Thermo Fisher Scientific,Gibco TM,目录号:25300062)
  12. 1×DPBS,不含钙,不含镁(Thermo Fisher Scientific,Gibco TM,目录号:14190-094)
  13. 1,4-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  14. 含有稳定剂(Sigma-Aldrich,目录号:H1009)的H 2 O中的30%(w / w)的过氧化氢溶液。
  15. 兴奋剂和抑制剂(这些是依赖于实验的)
  16. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  17. 氯化钾(KCl)(VWR,AnalaR NORMAPUR®,目录号:26764.298)
  18. 氯化钙二水合物(CaCl 2•2H 2 O)(Merck,目录号:102382)
  19. 氯化镁(MgCl 2)(Merck,目录号:105833025)
  20. D(+) - 无水葡萄糖(Merck,目录号:108337)
  21. EGTA(Sigma-Aldrich,目录号:E4378)
  22. 1M HEPES(Sigma-Aldrich,目录号:H7523)
  23. 林格缓冲液(0.25mM Ca2 +,pH7.4)(见食谱)

设备

  1. Z系列电脑计数器(Beckman Coulter,型号:6605699)和粒子计数器Z1(Beckman Coulter或任何其他计数装置)
  2. 培养箱,湿度和气体控制细胞培养
  3. Zeiss Axio Observer.Z1(Carl Zeiss,型号:Axio Observer.Z1)设置(图1)(孵育系统S包括Temp模块S,CO模块S,O 模块S,加热模块S)
  4. 镊子(,例如,样式Dumont Nr。7)
  5. 成像室和环插入(自制)和灌注系统(图2)


    图1. Zeiss Cell Observer.Z1设置温度,CO 2控制单元,气室和灌注系统。 :一种。分析电脑; B.具有40x油物镜和相应滤光器组的Cell Observer.Z1; C. Evolve 512 x 512 EM-CCD相机; D. CO 2供应装置; E. Pecon XL S1培养箱和控制模块; F. LED Colibri与相应的模块。 G.泵和灌注系统。
    注意:
    1. 对于HyPer测量,CFP / YFP滤波器是必不可少的,但具有相同特性的多频带滤波器立方体也是一个功能选项。
    2. 对于HyPer实验,我们使用波长为505 nm和420 nm的LED光源和相应的分束器。


      图2.带温度控制的成像室和安装模块:一种。自制的成像室(一)与灌注室塑料插入(二),与旋钮固定(三)。带有细胞的盖玻片连接到塑料插入物的下部,并且将12mm的小盖玻片连接到塑料插入物的上部以形成用于测量的小灌注通道。 B.成像室连接到灌注系统和显微镜的阶段(一)。灌注管(ii)连接到注射器上以便在测量过程中添加溶液,而第二灌注管(iii)连接到抽吸泵系统以除去废液。

软件

  1. Axiovision 4.6v(蔡司)具有快速采集功能和测量分析或类似的许可证

程序

  1. 第一天:细胞培养和播种
    该方案对于粘附细胞是示例性的,其可以用诸如Fugene HD的试剂转染。对于难以转染的细胞系,我们推荐替代转染方法(例如电穿孔核转染)。如果表达所需传感器的稳定细胞是可用的,则它们也可以用于本方案中描述的成像实验。
    1. 用相应的生长培养基培养细胞,直到达到约70%的汇合度。取出生长培养基,用5ml DPBS洗涤细胞一次,并在室温下将它们与1ml胰蛋白酶或1ml accutase(用于正常培养)一起温育来分离细胞。
    2. 将细胞悬浮在生长培养基中,用DPBS以100μl的比例稀释100μl,总体积为10ml。
      用Z1细胞计数器或血细胞计数器确定稀释液中的细胞浓度
    3. 将高压灭菌的玻璃盖玻片置于6孔板中,并在2ml生长培养基中的每个孔中接种400,000个细胞(在本实施例中为HEK293细胞)。将培养板置于加湿细胞培养箱(37℃,5%CO 2)中孵育过夜。

  2. 第2天:转染
    1. 从冰箱中取出Opti-MEM培养基和Fugene HD溶液,并在室温下平衡几分钟。
    2. 将100μlOpti-MEM培养基与4至10μlFugene HD溶液(根据生产商的方案)混合,加入建议量的质粒DNA(1μg/μl无内毒素储备溶液)(推荐1μg/孔,但最佳用量可以不同,取决于细胞类型和质粒)。将混合物上下吸移15次。
      注意:最佳的转染条件,如细胞密度,DNA量,DNA:Fugene HD比率可能需要对细胞进行优化行的选择。
    3. 在室温下等待15分钟,然后向每个孔中加入100μl转染混合物。
      注意:
      1. 如果您的细胞生长培养基含有抗生素,建议在转染之前将其改为不含抗生素的生长培养基,因为它们可能降低转染效率;否则,在转染混合物加入之前不需要改变生长培养基。
      2. 由于mitoHyPer和mitoSypHer具有相同的光谱特征,因此应该分别转染(在不同的孔中)。
    4. 将细胞在潮湿细胞培养箱(37℃,5%CO 2)中孵育。用新鲜的细胞生长培养基在6小时后改变转染孔中的培养基。将细胞保持在培养箱中,直到准备成像(37℃,5%CO 2),持续约24-48小时。

  3. 第3天或第4天:成像
    用Zeiss Cell Observer.Z1设置温度,CO 2控制单元,气室和灌注系统(图1)进行成像。
    1. 用一对精细的镊子轻轻地去除细胞覆盖的盖玻片(避免在盖玻片的中央成像区域中细胞的报废)。在灌注室塑料插入物(自制)的底部边缘添加Baysilone贴,并将其贴在盖玻片上(细胞朝上)。为了创建一个小的灌注通道,在塑料插入物的上部固定一个带有Baysilone贴的12毫米盖玻片。然后用旋钮固定塑料插入物(盖玻片),并将组装的腔室放入金属成像室(见图2A)。
      注意:如果只进行简单的实验,分析静息水平,然后加入饱和的H,使用标准成像室或玻璃底板可能就足够了,并通过用移液管小心添加试剂来刺激信号变化。但是对于更精确的实验来说,建议使用灌注系统进行多次添加或洗出实验。
    2. 将成像室放在显微镜载物台上,并将含有灌注管的溶液(避免系统中的空气)连接到室的相对两侧(如图2B所示)。用2毫升林格溶液(请参阅食谱)轻轻灌洗洗去分离的细胞。等待5分钟以达到CO 2(5%)和温度(37℃)平衡,然后再进行处理。
      注意:CO和温度由成像系统和相应的控制单元控制和监控。我们的灌注系统的一侧有一个注射器,可以手动使用林格溶液,另一侧使用抽吸泵来清除废物。
    3. 使用40倍的目标,搜索一个适当的视野,使您可以评估单独的,连接良好的细胞。设置LED强度以获得适当的信号,但不能太高以避免传感器的光漂白。优化曝光时间以获得良好的图像质量(背景上的信号),并将两个通道(420nm对505nm)的曝光时间比保持为所有实验的常数。根据所使用的细胞类型和可用的设备,这部分程序需要一些时间进行优化,因为光源和照相机可能会有所不同。
    4. 通过每1秒钟测量细胞中H 2 O 2的静止水平开始实验至少10秒,然后通过灌注系统添加刺激物质并记录直到信号稳定(或根据刺激方案)。应该优化每分钟的帧数和总的成像时间以获得适当的时间分辨率,而且还要避免光漂白。
      注意:根据科学问题和细胞类型,导致线粒体产生ROS的刺激物质是不同的。对于其他科学问题,只有静息的氧化还原水平(例如正常情况下的生理浓度)可能是有意义的。 >
    5. 在每次测量结束时,应加入单剂量的饱和H 2 O 2(例如1mM)作为阳性对照并确定传感器的最大强度(这可能需要校准系统)。为了检测完全减少的传感器的荧光强度(这将指示传感器在静息条件下是否已经被氧化并提供最小值用于校准),我们建议添加还原剂(例如,2mM DTT)在实验结束。
    6. 使用mitoSypHer传感器作为成像控制执行相同的成像过程,因为HyPer传感器可能会受到pH变化的影响。
      注意:如果使用HyPer传感器进行实验期间获得的信号(或比率变化)是由于氧化造成的,则在相同的实验条件下,SypHer信号不会发生任何变化。如果pH影响你的结果,你也会看到SypHer传感器的比例的变化。由于HyPer是一个pH依赖型传感器,为了讨论有关氧化还原变化的数据,这种控制是强制性的。

数据分析

使用Axiovision软件进行分析

  1. 背景更正
    应该通过从目标(基于单元的)ROI减去背景ROI中的强度值来执行背景校正(图3)。


    图3.表达mitoHyPer的HEK293细胞的分析实例(也参见图4)A.合并的图像(420nm绿色,505nm红色)。 B.分析的示例性介绍。红色圆圈表示无细胞区域的背景ROI,而一些细胞的边界用白色徒手绘制的ROI标记进行分析。

  2. 比率分析
    比例动力学曲线是用下面的公式生成的:



    使用Axiovision软件。
    注意:数据分析可以使用来自其他供应商的不同软件来执行。 HyPer比率的基本计算也可以使用开源软件来完成,例如ImageJ( https ://imagej.nih.gov )。
    数据通常以平均值±SEM(或SD)表示,并用双侧Student's检验显着性。对于每个条件,至少有三个实验应该进行适当的重复。
  3. 以下是代表性图像(A),放大单细胞(B),图表总结(C)和统计分析(D)HEK293细胞在H 2 O 2>和DTT添加(图4)。
    注意:如果探头在测量过程中被完全氧化,并且无法响应饱和H 2

    图4.表达线粒体H 2 O 2传感器mitoHyPer的HEK293细胞的示例性ROS测量。在成像之前48小时,使用基于Fugene HD的溶液,用mitoHyPer传感器转染HEK293细胞。首先用50μM和500μMH 2 O 2滴定细胞用于探针的氧化。在H 2 O 2 O 2清除之后,用2mM DTT滴定细胞以还原探针。荧光强度比的变化表示为合并图像(420nm绿色,505nm红色)。如(A)所示,加入H 2 O 2 2引起探针氧化并增加信号比率,而添加DTT降低信号比率。 (B)中放大显示(A)中的指示区域。由于线粒体H 2 O 2在细胞静息状态下产生,所以探针可以被组成性产生的ROS部分氧化,并且可以通过膜渗透还原剂还原作为DTT。 (D)中的(C)和(D)中的统计分析(平均值±SEM,n = 17)中示出对应于(A)和(B)中的图像的时间进程。比例尺= 10微米。

食谱

  1. 林格缓冲液(0.25mM Ca 2 +,pH7.4)
    155 mM NaCl
    4.5 mM KCl
    10 mM葡萄糖
    5毫米HEPES
    2.75mM MgCl 2•/ 2 0.25 mM CaCl 2 2/2

致谢

德国研究基金会(DFG)通过SFB1190项目17,SFB1027项目C4和BO3643 / 3-2研究资助(均为IB)支持这项工作。作者声明没有利益冲突或竞争的财务利益。

参考

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, X., Gibhardt, C. S., Cappello, S., Zimmermann, K. M., Vultur, A. and Bogeski, I. (2018). Measuring Mitochondrial ROS in Mammalian Cells with a Genetically Encoded Protein Sensor. Bio-protocol 8(2): e2705. DOI: 10.21769/BioProtoc.2705.
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