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Feb 2020

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Monitoring Changes in the Oxidizing Milieu in the Endoplasmic Reticulum of Mammalian Cells Using HyPerER
利用HyPerER监测哺乳动物细胞内质网氧化环境的变化   

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Abstract

The production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress are tightly linked. The generation of ROS can be both the cause and a consequence of ER stress pathways, and an increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. For the assessment of modulators of ER luminal ROS generation and for mechanistic studies, methods to monitor changes in ER reduction-oxidation (redox) states in a time-resolved and organelle-specific manner are needed. This has been greatly facilitated by the development of genetically encoded fluorescent probes, which can be targeted to different subcellular locations by specific amino acid extensions. One of these probes is the yellow fluorescent protein-based redox biosensor, HyPer. Here, we provide a protocol for the time-resolved monitoring of the oxidizing milieu in the ER of adherent mammalian cells using the ratiometric sensor, HyPerER, which is specifically targeted to the ER lumen.

Keywords: Endoplasmic reticulum (内质网), Hydrogen peroxide (过氧化氢), HyPer (HyPer), Redox (氧化还原), Oxidative stress (氧化应激), Fluorescence microscopy (荧光显微镜)

Background

The endoplasmic reticulum (ER) plays key roles in essential functions including protein folding and maturation in the secretory pathway, lipid metabolism, hormone synthesis, and detoxification of reactive metabolites (Bock and Kohle, 2009; Chen and Cubillos-Ruiz, 2020; Erdbrugger and Frohlich, 2020; Morishita and Arvan, 2020). Research on the ER has attracted increasing interest in recent years, mainly due to the discovery of the unfolded protein response (UPR) signaling pathway, which is triggered by diverse forms of protein folding stress in the ER. The physical contact sites of the ER with other cell organelles and their involvement in cellular communication networks establish the ER as a multifaceted regulator of cell signaling. The relationship between ER stress and oxidative injury has been extensively investigated; however, the origin of ER stress-induced ROS production remains unclear (Appenzeller-Herzog, 2011), and tools to detect xenobiotics that enhance ROS in the ER are limited.


To understand the mechanisms of oxidative insults, specific tools are required to quantitate and describe ER redox conditions. Genetically encoded sensors to quantitate the oxidative status of the many redox couples present in the ER have proven useful. Changes in the amount of H2O2 in the cytoplasm can be monitored using the fluorescent probe, HyPer (Belousov et al., 2006). The HyPer sensor was constructed by inserting a circularly permutated yellow fluorescent protein (YFP) into the regulatory domain of the bacterial H2O2-sensing protein, OxyR. Importantly, HyPer was shown to selectively detect H2O2 over superoxide, peroxinitrite, nitric oxide, and oxidized glutathione in the cytosol. Upon oxidation of the cysteine corresponding to Cys199 of OxyR, the sensor protein HyPer undergoes a conformational change. HyPer has two excitation peaks at 420 nm and 500 nm, and one emission peak at 516 nm. Upon transition from the reduced to the oxidized state, the peak at 420 nm decreases and the peak at 500 nm increases, thus allowing ratiometric measurement of H2O2.


Here, we describe a detailed protocol for the real-time imaging and monitoring of the oxidizing milieu in the ER using the HyPerER sensor, which is targeted specifically to the ER lumen (Enyedi et al., 2010; Malinouski et al., 2011) by the addition of an N-terminal ER-targeting sequence and a C-terminal ER-retrieval signal (KDEL). It must be noted that the ER-targeted HyPerER, unlike cytosolic HyPer, is not specific for H2O2 but rather reflects the oxidative milieu within the ER (Mehmeti et al., 2012). The approach can be used in different cellular systems with basic understanding of live cell imaging and fluorescence microscopy (an example is shown in Figure 1), whereby data analysis is dependent on the available software. A limitation of the original HyPer sensors is their sensitivity to pH changes. To overcome this limitation, a very recent study introduced a new generation HyPer probe, HyPer7, which is pH-resistant yet remains ultra-sensitive to changes in H2O2 (Pak et al., 2020). Monitoring H2O2 specifically in the ER remains challenging, and most of the probes available to date have not been used in this very special cellular compartment.


Materials and Reagents

  1. Materials

    1. Pipette tips

    2. Glass-bottomed dishes (such as MatTek, catalog number: P35G-1.5-20-C; IBIDI µ-Dish 35 mm, high Glass Bottom, catalog number: 81158; Sarstedt lumox dish 35, catalog number: 94.6077.331; Nunc Glass Bottom Dishes, catalog number: 150680)

    3. 1.5-ml tubes

    4. 50-ml conical centrifugation tubes


  2. Reagents

    1. HeLa cells (ATCC, catalog number: CCL-2)

    2. Dulbecco’s Modified Eagle’s Medium (DMEM) – high glucose (Sigma-Aldrich, catalog number: D5796)

    3. Fetal bovine serum, FBS (South America) (Biowest, catalog number: S1810)

    4. Penicillin/streptomycin 100× (BioConcept-Amimed, catalog number: 4-01F00-H)

    5. Trypsin-EDTA solution 10× (Sigma-Aldrich, catalog number: T4174-100ML)

    6. OptiMEM-I (Gibco, catalog number: 51985026)

    7. FuGENE HD (Promega, catalog number: E2311)

    8. HEPES (PanReac AppliChem, catalog number: A1069)

    9. CaCl2·2H2O (Merck, catalog number: 1.02382.0500)

    10. KCl (Merck, catalog number: 1.04936.1000)

    11. MgCl2·6H2O (Fluka, catalog number: 63064)

    12. NaCl (PanReac AppliChem, catalog number: A2942)

    13. DTT (PanReac AppliChem, catalog number: A1101)

    14. H2O2 solution (Sigma-Aldrich, catalog number: 95321)

    15. pCMV/myc/ER/GFP HyPerER (Enyedi et al., 2010) (Kind gift from Dr. Miklos Geiszt, Semmelweis University, Budapest, Hungary)

    16. Stimulants and inhibitors (experiment-dependent), e.g., thapsigargin (EMD Millipore, catalog number: 586005)

    17. HEPES Imaging Buffer (1 L) (see Recipes)

Equipment

  1. Pipettes

  2. Casy cell counter (Omni Life Science) or hemocytometer

  3. Heat block for 50-ml conical tubes

  4. Inverted microscope; we use an Olympus Fluoview3000 laser scanning microscope

  5. 60× Objective UPLSAPO60XS2 Universal Plan Super Apochromat silicone immersion objective N.A. 1.3 (N5203000)

  6. For excitation, we use an Olympus FVL-LAS405-LX50 Laser 405 nm and FVL-LAS488-LS20 Laser 488 nm (somewhat below the excitation peaks at 420 nm and 500 nm, respectively)

    Note: Monitoring of HyPer probes does not require confocality! A fluorescence microscope with suitable filter sets for excitation and emission is sufficient.

    Excitation maximum of HyPer: 500 nm

    Emission maximum of HyPer: 516 nm

  7. Climate control unit; we use the Olympus CellVivo incubator system for IX83 (E0439957)

    Note: Live cell experiments should be performed under optimal environmental conditions; the minimal requirement is a temperature controller to maintain the optimal temperature of 37°C; for long-term experiments, e.g., longer than 30 min, an additional CO2 supply will be needed.

Software

  1. FV300 (Olympus)

  2. Excel (Microsoft)

Procedure

  1. Cell culture and seeding

    Cells should be cultured in their corresponding growth medium until they reach a confluence of around 70%.

    1. Wash the cells with 10 ml 1× PBS.

    2. Detach the cells with 2 ml pre-warmed (37°C) 1× trypsin (0.5 g/L) at room temperature for 4 min and resuspend in 8 ml complete growth medium.

    3. Determine the number of cells in the suspension using a Casy cell counter or hemocytometer.

    4. Seed 30,000 cells in 400 µl complete growth medium to the center well of a Matek glass-bottomed dish.

      Cell density may depend on the cell type and transfection method used.

    5. Place the dish in a humidified cell culture incubator (37°C, 5% CO2) and incubate overnight.


  2. Transfection

    1. Mix 25 µl Opti-MEM medium with 0.5 µg HyPerER plasmid and 1.5 µl Fugene® HD solution (according to the manufacturer’s protocol).

    2. Incubate the mixture for 15 min at room temperature and add dropwise to the cells.

    3. Add 2.6 ml complete growth medium approximately 6 h after transfection.

    4. Incubate the cells at 37°C for 24-48 h.

      Note: The optimal transfection conditions, e.g., cell density, DNA amount, and DNA:Fugene® HD ratio, will require optimization depending on the cell line of choice.

  3. Imaging

    Imaging is performed on an Olympus Fluoview 3000 laser-scanning microscope with a temperature- and CO2-controlling unit. Samples are excited sequentially using the 405 nm and 488 nm lasers. Emission is recorded in a window from 500 to 600 nm.

    Note: There is only one emission window for both excitation wavelengths!

    1. Take out the cell culture dish from the incubator and remove culture medium with a pipette.

    2. Carefully wash the dish twice with 1 ml pre-warmed HEPES Imaging Buffer.

    3. Add 1 ml HEPES Imaging Buffer and place the dish on the microscope stage.

    4. Search for a field of view that contains several healthy-looking cells with sufficient YFP fluorescence signal.

    5. Optimize the voltage of the photomultiplier tube to obtain good image quality (signal-to-noise) for both channels.

    6. Measure the baseline oxidative status of HyPerER in the cells every 20 s for at least 5 min, then carefully add stimulating substances, such as thapsigargin (final concentration 1 µM), at double working concentration in 1 ml HEPES Imaging Buffer with a pipette and record until the signal stabilizes (or according to the stimulation protocol). Make sure to complete the addition within the 20-s time window between two image acquisitions. The speed of the acquisition and the total imaging time should be optimized to achieve proper temporal resolution but also to avoid photobleaching.

      Conclude each measurement by the addition of a single dose of saturating H2O2 (100 µM final concentration in 1 ml HEPES Imaging Buffer) as a positive control, carefully applied with a pipette. An example in HeLa cells is shown in Figure 1.

Data analysis

Analysis using the FV3000 software:

  1. Define the regions of interest (ROI) in your sample and select a suitable area to place a background ROI.

  2. Measure the raw emission intensity within these ROIs.

  3. Export the raw intensity values from the software as .csv files.

  4. Open the .csv files in Microsoft Excel. You will obtain two emission intensity values, determined at 516 nm, for each time point measured for every ROI, one for excitation at 488 nm and one for excitation at 405 nm. Perform the following calculation:

    Subtract the intensity values of the background ROI obtained at 405-nm excitation from the target ROI measured at 405 nm.

    Subtract the intensity values of the background ROI obtained at 488-nm excitation from the target ROI measured at 488 nm.

    Calculate the fluorescence ratio using the following formula (for an example, see Figure 1):




    Figure 1. Real-time monitoring of ER redox changes. HeLa cells were transfected with the HyPerER sensor using Fugene® HD. At 48 h post-transfection, the cells were prepared for imaging. Fluorescence ratio changes were monitored over time. A. Each trace corresponds to the data recorded from one cell. The cells were treated with 1 µM thapsigargin (TG), which reduces the ER environment by facilitating the influx of reduced glutathione (Lizak et al., 2020), followed by the application of 100 µM H2O2, which leads to re-oxidation. B. Exemplary fluorescence pictures in the 405-nm or 488-nm channel and the corresponding ratiometric images. Scale bar = 10 µm. The pseudocolored images represent the ratio R, obtained by dividing the values measured at 488-nm excitation and 516-nm emission after background subtraction by the values measured at 405-nm excitation and 516-nm emission. A darker color indicates a lower ratio and hence less H2O2; a lighter color represents a higher ratio and hence more H2O2.


Recipes

  1. HEPES Imaging Buffer (1 L)

    1 M HEPES solution (sterile-filtered) 20 ml; final concentration 20 mM

    1 M KCl solution (sterile-filtered) 5 ml; final concentration 5 mM

    1 M CaCl2 solution (sterile-filtered) 1.8 ml; final concentration 1.8 mM

    1 M MgCl2 solution (sterile-filtered) 1 ml; final concentration 1 mM

    1 M NaCl solution (sterile-filtered) 130 ml; final concentration 130 mM

    Fill to 1 L with ddH2O and adjust pH to 7.4

Acknowledgments

This protocol was adapted from Lizak et al. (2020). Funding by the Swiss National Science Foundation (SNSF, 31003A-179400) and the University of Basel is gratefully acknowledged.

Competing interests

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Appenzeller-Herzog, C. (2011). Glutathione- and non-glutathione-based oxidant control in the endoplasmic reticulum. J Cell Sci 124(Pt 6): 847-855.
  2. Belousov, V. V., Fradkov, A. F., Lukyanov, K. A., Staroverov, D. B., Shakhbazov, K. S., Terskikh, A. V. and Lukyanov, S. (2006). Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3(4): 281-286.
  3. Bock, K. W. and Kohle, C. (2009). Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: advances and open questions. Biochem Pharmacol 77(9): 1458-1465.
  4. Chen, X. and Cubillos-Ruiz, J. R. (2021). Endoplasmic reticulum stress signals in the tumour and its microenvironment. Nat Rev Cancer 21(2): 71-88.
  5. Enyedi, B., Varnai, P. and Geiszt, M. (2010). Redox state of the endoplasmic reticulum is controlled by Ero1L-alpha and intraluminal calcium. Antioxid Redox Signal 13(6): 721-729.
  6. Erdbrugger, P. and Frohlich, F. (2020). The role of very long chain fatty acids in yeast physiology and human diseases. Biol Chem 402(1): 25-38.
  7. Lizak, B., Birk, J., Zana, M., Kosztyi, G., Kratschmar, D. V., Odermatt, A., Zimmermann, R., Geiszt, M., Appenzeller-Herzog, C. and Banhegyi, G. (2020). Ca2+ mobilization-dependent reduction of the endoplasmic reticulum lumen is due to influx of cytosolic glutathione. BMC Biol 18(1): 19.
  8. Malinouski, M., Zhou, Y., Belousov, V. V., Hatfield, D. L. and Gladyshev, V. N. (2011). Hydrogen peroxide probes directed to different cellular compartments. PLoS One 6(1): e14564.
  9. Mehmeti, I., Lortz, S. and Lenzen, S. (2012). The H2O2-sensitive HyPer protein targeted to the endoplasmic reticulum as a mirror of the oxidizing thiol-disulfide milieu. Free Radic Biol Med 53(7): 1451-1458.
  10. Morishita, Y. and Arvan, P. (2020). Lessons from animal models of endocrine disorders caused by defects of protein folding in the secretory pathway. Mol Cell Endocrinol 499: 110613.
  11. Pak, V. V., Ezerina, D., Lyublinskaya, O. G., Pedre, B., Tyurin-Kuzmin, P. A., Mishina, N. M., Thauvin, M., Young, D., Wahni, K., Martinez Gache, S. A., Demidovich, A. D., Ermakova, Y. G., Maslova, Y. D., Shokhina, A. G., Eroglu, E., Bilan, D. S., Bogeski, I., Michel, T., Vriz, S., Messens, J. and Belousov, V. V. (2020). Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function. Cell Metab 31(3): 642-653 e646.

简介

[摘要]在P反应性oxyg的roduction烯物种(ROS)和内质网(ER)应力紧紧连接。ROS的生成既可以是所述原因和一个ER的结果应激途径,以及越来越多的人的疾病的响应于ER的特征在于组织萎缩 压力和氧化损伤。为了评估 ER 腔内 ROS 生成的调节剂和机制研究,需要以时间分辨和细胞器特异性方式监测 ER 还原-氧化(氧化还原)状态变化的方法。遗传编码荧光探针的开发极大地促进了这一点,该探针可以通过特定的氨基酸延伸靶向不同的亚细胞位置。一个这些探针的是黄色荧光蛋白质基氧化还原生物传感器,数据HyPer 。在这里,我们提供用于时间分辨监测使用贴壁哺乳动物细胞的ER的氧化环境的协议比例传感器,HyPerER ,其特异性地靶向内质网腔。


[背景]所述的内质网(ER)起着基本功能键的作用,包括蛋白折叠和成熟在秒retory途径,脂质代谢,激素合成,和反应性代谢物的解毒(博克和Kohle,2009;陈和Cubillos-Ruiz的, 2020;Erdbrugger和Frohlich,2020;Morishita和Arvan,2020) 。研究ER已在吸引了越来越多的关注最近几年,主要是由于其被触发未折叠蛋白反应(UPR)信号通路的发现通过在ER折叠应激蛋白的不同形式。ER与其他细胞器的物理接触位点及其参与细胞通信网络将 ER 建立为细胞信号的多方面调节器。内质网应激与氧化损伤之间的关系已被广泛研究。然而,内质网应激诱导的 ROS 产生的起源仍不清楚(Appenzeller-Herzog,2011),并且检测增强内质网中 ROS 的异生物质的工具是有限的。

牛逼Ø理解氧化损伤的机制,具体工具即可孔定量泰特和描述ER氧化还原条件。基因编码的传感器孔定量泰特存在于ER的许多氧化还原电对的氧化状态已被证明是有用的。在H量的变化2 Ó 2在细胞质中可以使用荧光探针来监测,数据HyPer (贝洛索夫等人,2006) 。所述数据HyPer传感器通过插入一个环行排列的黄色荧光蛋白(YFP)到细菌H的调节结构域构造2 ö 2 -sensing蛋白,OxyR 。重要的是,数据HyPer显示出选择性地检测ħ 2 ö 2以上超氧化物,peroxinitrite ,一氧化氮,并在细胞质中的氧化型谷胱甘肽。在氧化对应于OxyR 的Cys199 的半胱氨酸时,传感器蛋白HyPer会发生构象变化。HyPer在 420 nm 和 500 nm 处有两个激发峰,在 516 nm 处有一个发射峰。在从还原态转变为氧化态时,420 nm 处的峰值减小而 500 nm 处的峰值增加,从而允许对 H 2 O 2进行比率测量。

在这里,我们描述了一个详细的协议的实时成像,并使用在ER监测氧化性环境的HyPerER传感器,其特异性地靶向内质网腔(Enyedi等人,2010; Malinouski 。等人,2011)通过所述添加N端ER的-靶向序列和C端ER -检索信号(KDEL)。它必须指出的是,ER-针对性HyPerER ,不像胞浆数据HyPer ,是不特定用于h 2 Ø 2 ,而是反映了ER内的氧化环境(梅赫梅蒂等,2012) 。该方法可用于不同的细胞系统,对活细胞成像和荧光显微镜有基本了解(图1 中显示了一个示例),其中数据分析取决于可用的软件。原始HyPer传感器的一个限制是它们对 pH 变化的敏感性。为了克服这一限制,最近的一项研究引入了新一代HyPer探针 HyPer7,它具有抗pH 值,但仍然对 H 2 O 2 的变化非常敏感(Pak等人,2020 年)。监测^ h 2 Ø 2特别是在急诊室仍然具有挑战性和最可用探针迄今尚未在这个非常特殊的细胞室中使用。

关键字:内质网, 过氧化氢, HyPer, 氧化还原, 氧化应激, 荧光显微镜

材料和试剂
 
材质小号
移液器吸头
玻璃-底部ED菜(如马蒂克,目录号:P35G-1.5-20-C; IBIDIμ-盘35毫米,高玻璃底,目录号:81158; Sarstedt的lumox盘35,目录号:94.6077.331; Nunc公司玻璃底盘,目录号:150680)
1.5 -毫升管
50 - ml 锥形离心管
 
试剂
HeLa细胞(ATCC,目录号:CCL-2)
Dulbecco 改良 Eagle 培养基(DMEM)-高葡萄糖(Sigma - Aldrich,目录号:D5796)
胎儿b羊小号erum,FBS(南美洲)(Biowest ,目录号:S1810)
青霉素/链霉素100 × (BioConcept-Amimed ,目录号:4 -01F00-H )
胰蛋白酶- EDTA小号olution 10 × (西格玛- Aldrich公司,目录号:T4174-100ML)
OptiMEM -I(Gibco,目录号:51985026)
FuGENE HD(Promega,目录号:E2311)
HEPES(PanReac AppliChem,目录号:A1069)
氯化钙2· 2H 2 O(默克,目录号:1.02382.0500)
KCl (默克,目录号:1.04936.1000)
MgCl 2 · 6H 2 O(Fluka ,目录号:63064)
NaCl(PanReac AppliChem,目录号:A2942)
DTT(PanReac AppliChem,目录号:A1101)
H 2 O 2溶液(Sigma-Aldrich,目录号:95321)
pCMV / myc /ER/GFP HyPerER (Enyedi等人,2010 年)(来自匈牙利布达佩斯 Semmelweis 大学Miklos Geiszt博士的善意礼物)
兴奋剂和抑制剂(实验依赖型),例如,毒胡萝卜素(密理博,目录号:586005)
HEPES 成像缓冲液(1 L)(见配方)
 
设备
 
移液器
凯西Ç ELL计数器(全生命科学)或血球
热块50 -毫升锥形管中
倒置显微镜;我们使用奥林巴斯 Fluoview3000 激光扫描显微镜
60 ×物镜 UPLSAPO60XS2 Universal Plan Super Apochromat 硅胶浸没物镜 NA 1.3 (N5203000)
对于激发,我们使用一个(分别稍微低于在420nm和500nm,激发峰)奥林巴斯FVL-LAS405-LX50激光405nm和FVL-LAS488-LS20激光488nm的
注:中号onitoring的数据HyPer探头不需要共焦!带有用于激发和发射的合适滤光片组的荧光显微镜就足够了。
HyPer 的最大激发波长:500 nm
HyPer 的最大发射波长:516 nm
气候控制单元;我们使用适用于 IX83的Olympus CellVivo培养箱系统 (E0439957)
注意:活细胞实验应在最佳环境条件下进行;最低要求是温度控制器,以保持 37°C 的最佳温度;长-术语实验,例如,长超过30分钟,一个额外的CO 2 ,将需要供应。
 
软件
 
FV300(奥林巴斯)
Excel(微软)
 
程序
 
细胞培养和接种
细胞应在其相应的生长培养基中培养,直到它们达到约 70%的汇合。
洗的细胞用10ml 1 × PBS 。
用 2 ml 预热 (37°C) 1 ×胰蛋白酶 (0.5 g/L) 在室温下分离细胞4 分钟,然后重悬在 8 ml 完全生长培养基中。
使用Casy细胞计数器或血细胞计数器确定悬浮液中的细胞数。
种子30 ,000个细胞在400μl的完全生长介质的中心孔的Matek的玻璃-底部版盘。
细胞密度米AY取决于所使用的细胞类型和转染方法。
将培养皿置于加湿的细胞培养箱(37°C,5% CO 2 )中并孵育过夜。
 
转染
用0.5μg混合25微升的Opti-MEM培养基HyPerER p lasmid和1.5微升Fugene转® (根据制造商的方案)HD溶液。
孵育该混合物在室温下15分钟,并逐滴添加至所述细胞。
转染后约 6 小时加入2.6 ml 完全培养基。
孵育细胞在37℃培养24 - 48小时。
注:最佳转染条件下,例如,细胞密度,DNA量,和DNA:Fugene转® HD比,将需要优化是否取决于荷兰国际集团上所选择的细胞系。
 
成像
成像在Olympus进行Fluoview共3000激光扫描显微镜一个温度-和CO 2 -控制单元。样品依次使用 405 nm 和 488 nm 激光s激发。发射被记录在窗口从500至600nm 。
注:牛逼这里只有两个激发波长一个发射窗口!
取出来的细胞培养皿用吸管培养箱和移除培养基。
用 1 ml 预热的 HEPES 成像缓冲液小心地将盘子洗两次冰。
加入1 ml HEPES成像缓冲器,并放置在显微镜载物台上的菜。
搜索包含几个健康的视野-寻找细胞足够的YFP荧光信号。
优化光电倍增管的电压,以获得良好的图像质量(信号-到-噪声)为两个通道。
每 20 秒测量一次细胞中HyPerER的基线氧化状态至少 5 分钟,然后小心地加入刺激性物质,如毒胡萝卜素(终浓度 1 µM),在 1 ml HEPES 成像缓冲液中以双倍工作浓度用移液管并记录直到信号稳定(或根据刺激方案)。确保完成20以内的加法-的两个图像采集之间的时间窗口。采集速度和总成像时间应进行优化,以实现适当的时间分辨率,但也避免光漂白。
通过添加单剂量的饱和 H 2 O 2 (100 µM 最终浓度在 1 ml HEPES 成像缓冲液中)作为阳性对照,用移液器小心地应用,结束每次测量。图 1 显示了 HeLa 细胞中的一个示例。
 
数据分析
 
使用FV3000 软件进行分析:
定义了你的样品中感兴趣区域(ROI),并选择合适的区域来放置背景的投资回报率。
测量这些 ROI 内的原始发射强度。
将软件中的原始强度值导出为 .csv文件。
打开该.csv文件小号在Microsoft Excel中。你将获得两个发射强度值,在516处确定的,为电子ACH为每个ROI,一个用于在488nm处激发和一个用于在405nm激发测量时间点。执行以下计算:
减去ROI在405获得的背景的强度值-从在405nm处测得的靶ROI nm激发。
减去ROI在488获得的背景的强度值-从在488nm处测得的靶ROI nm激发。
 
使用以下公式计算荧光比(例如参见图 1):
 
 
图 1. ER 氧化还原变化的实时监测。使用Fugene ® HD用HyPerER传感器转染HeLa 细胞。在48小时后-转染,用于成像,制备细胞。随时间监测荧光比率变化。A. 每条轨迹对应一个单元格记录的数据。细胞用 1 µM毒胡萝卜素(TG) 处理,通过促进还原型谷胱甘肽的流入来减少ER 环境(Lizak等人,2020 年),然后应用 100 µM H 2 O 2 ,这导致重新氧化。B. 405 - nm 或 488 - nm 通道中的示例荧光图片和相应的比例图像。比例尺 = 10 µm。所述伪彩图像表示的比率R ,获得通过将在488nm测得的值- nm激发和516 -后通过在405测得的值减去背景nm发射- nm激发和516 -发射波长。甲d arker颜色表示一个更低的比率,并因此较少ħ 2 ö 2 ; 一个较浅的颜色表示较高的比率和因此多个H 2 ö 2 。
 
食谱
 
HEPES 成像缓冲液 (1 L)
的1M HEPES溶液(无菌- FILT ER ED )20毫升; 终浓度 20 mM                                                                                                               
的1M的KCl溶液(无菌- FILT ë红色)5毫升; 终浓度 5 mM                           
的1M的CaCl 2溶液(无菌- FILT ë红色)1.8毫升; 终浓度 1.8 mM                                         
的1M的MgCl 2溶液(无菌- FILT ER ED )1毫升; 终浓度 1 mM             
的1M NaCl溶液(无菌- FILT ER ED )130毫升; 终浓度 130 mM             
用 ddH 2 O填充至 1 L并将 pH 值调节至 7.4
 
致谢
 
该协议改编自Lizak等人。(2020 年)。非常感谢瑞士国家科学基金会 (SNSF, 31003A-179400) 和巴塞尔大学的资助。
 
利益争夺
 
作者声明个是研究在没有可能被视为潜在的利益冲突的任何商业或财务关系进行的。
 
参考
 
Appenzeller-Herzog, C. (2011)。内质网中基于谷胱甘肽和非谷胱甘肽的氧化剂控制。J Cell Sci 124(第 6 篇):847-855。
Belousov, VV, Fradkov, AF, Lukyanov, KA, Staroverov, DB, Shakhbazov, KS, Terskikh, AV 和 Lukyanov, S. (2006)。细胞内过氧化氢的基因编码荧光指示剂。Nat 方法3(4):281-286。
Bock, KW 和 Kohle, C. (2009)。内质网膜中寡聚 UDP-葡萄糖醛酸转移酶的拓扑方面:进展和开放性问题。生化药理学77(9):1458-1465。
Chen, X. 和 Cubillos-Ruiz, JR (2021)。肿瘤及其微环境中的内质网应激信号。Nat Rev Cancer 21(2):71-88。
Enyedi ,B.,Varnai ,P。和Geiszt ,M。(2010)。内质网的氧化还原状态受 Ero1L-α 和腔内钙的控制。抗氧化氧化还原信号13(6):721-729。
Erdbrugger , P. 和 Frohlich, F. (2020)。超长链脂肪酸在酵母生理学和人类疾病中的作用。生物化学402(1):25-38。
Lizak, B., Birk, J., Zana, M., Kosztyi, G., Kratschmar, DV, Odermatt, A., Zimmermann, R., Geiszt, M., Appenzeller-Herzog, C. 和 Banhegyi, G. (2020)。Ca 2+动员依赖的内质网腔减少是由于胞质谷胱甘肽的流入。BMC 生物学18(1):19。
Malinouski , M., Zhou, Y., Belousov , VV, Hatfield, DL 和Gladyshev , VN (2011)。过氧化氢探针针对不同的细胞隔室。PLoS一6(1):e14564。
Mehmeti, I.、Lortz, S. 和 Lenzen, S. (2012)。H 2 O 2敏感的HyPer 蛋白靶向内质网,作为氧化硫醇-二硫化物环境的镜子。免费拉迪奇生物学医学53(7):1451-1458。
Morishita , Y. 和Arvan , P. (2020)。由分泌途径中蛋白质折叠缺陷引起的内分泌失调动物模型的教训。分子细胞内分泌499:110613。
Pak, VV, Ezerina , D., Lyublinskaya , OG, Pedre , B., Tyurin-Kuzmin , PA, Mishina , NM, Thauvin , M., Young, D., Wahni , K., Martinez Gache , SA, Demidovich , AD, Ermakova , YG, Maslova , YD, Shokhina , AG , Eroglu , E., Bilan , DS, Bogeski , I., Michel, T., Vriz , S., Messens , J. 和Belousov , VV (2020)。过氧化氢的超灵敏基因编码指示器可识别氧化剂在细胞迁移和线粒体功能中的作用。细胞代谢31(3):642-653 e646。
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引用:Birk, J., Lizak, B., Appenzeller-Herzog, C. and Odermatt, A. (2021). Monitoring Changes in the Oxidizing Milieu in the Endoplasmic Reticulum of Mammalian Cells Using HyPerER. Bio-protocol 11(13): e4076. DOI: 10.21769/BioProtoc.4076.
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