【原】2016-Nature Protocols-Comprehensive analysis of mitochondrial permeability transition pore activity

2016-Nature Protocols-Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques

基于荧光成像技术的活细胞线粒体通透性过渡孔活性综合分析

Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques

基于荧光成像技术的活细胞线粒体通透性过渡孔活性综合分析

Massimo Bonora1,3, Claudia Morganti1,3, Giampaolo Morciano1,3, Carlotta Giorgi1, Mariusz R Wieckowski2

& Paolo Pinton1

1Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy. 2Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland. 3These authors contributed equally to this work. Correspondence should be addressed to P.P. ([e-mail]).

1费拉拉大学形态学、外科和实验医学系，病理学、肿瘤学和实验生物学教研室，高级治疗技术实验室(LTTA)，意大利费拉拉这些作者对这项工作贡献相同。信件请寄P.P. ([e-mail])。

Mitochondrial permeability transition (mPT) refers to a sudden increase in the permeability of the inner mitochondrial membrane. long-term studies of mPT revealed that this phenomenon has a critical role in multiple pathophysiological processes. mPT is mediated by the opening of a complex termed the mPT pore (mPTP), which is responsible for the osmotic influx of water into the mitochondrial matrix, resulting in swelling of mitochondria and dissipation of the mitochondrial membrane potential. Here we provide three independent optimized protocols for monitoring mPT in living cells: (i) measurement using a calcein cobalt technique, (ii) measurement of the mPTP-dependent alteration of the mitochondrial membrane potential, and (iii) measurement of mitochondrial swelling. these procedures can easily be modified and adapted to different cell types. cell culture and preparation of the samples are estimated to take ~1 d for methods (i) and (ii), and ~3 d for method (iii). the entire experiment, including analyses, takes ~2 h.

线粒体通透性转换(mPT)指线粒体内膜的通透性突然增加。对mPT的长期研究表明，这种现象在多种病理生理过程中起关键作用。mPT由称为mPT孔(mPTP)的复合物的开放介导，mPT孔负责水渗透流入线粒体基质，导致线粒体肿胀和线粒体膜电位耗散。在此，我们提供了三种用于监测活细胞中mPT的独立优化方案:(I)使用钙黄绿素钴技术进行测量，(ii)测量依赖于mPTP的线粒体膜电位改变，(iii)测量线粒体肿胀。这些程序可以容易地修改和适应不同的细胞类型。估计方法(I)和(ii)的细胞培养和样品制备需要~1 d，方法(iii)需要~3 d。整个实验，包括分析，需要大约2小时。

IntroDuctIon

mPT is believed to occur as a result of the opening of a nonspecific high-conductance channel in the inner mitochondrial membrane, which has become known as the mPTP. The mPTP may exist in low- and high-conductance modes1. Its low-conductance state is characterized by very limited permeability (cutoff, <300 Da), which permits the diffusion of small ions such as H+, Ca2+, and K+ but does not trigger detectable mitochondrial swelling. Alternatively, its high-conductance state (1-1.3 nS) allows the free movement of molecules with a molecular mass up to 1.5 kDa across the inner mitochondrial membrane and results in mitochondrial matrix swelling2.

mPT被认为是由于线粒体内膜中一个非特异性高电导通道的开放而发生的，该通道被称为mPTP。mPTP可能存在于低电导和高电导模式1中。其低电导状态的特征是渗透性非常有限(界限值，< 300 Da)，允许H+、Ca2+和K+等小离子扩散，但不会引发可检测到的线粒体肿胀。或者，其高电导状态(1-1.3 nS)允许分子量高达1.5 kDa的分子自由穿过线粒体内膜，从而导致线粒体基质肿胀2。

Accumulating evidence supports the speculation that the mPTP is related to mitochondrial ATP synthase3 7. Our hypothesis, which was validated using the protocol described herein, indicates that a key element of the mPTP core is the c-subunit of mitochondrial ATP synthase8,9. This conclusion was further supported by the observations of other groups10,11. Interestingly, this model does not exclude the dependence and regulation of mPT by regulatory proteins previously associated with the mPTP.

越来越多的证据支持mPTP与线粒体ATP合酶3 7相关的推测。使用本文所述方案验证的我们的假设表明，mPTP核心的一个关键要素是线粒体ATP合酶的c-亚基8，9。其他小组的观察结果进一步支持了这一结论10，11。有趣的是，该模型并未排除之前与mPTP相关的调节蛋白对mPT的依赖和调节。

Advantages and disadvantages of monitoring mPT in isolated mitochondria or intact cells

Numerous methods are available to monitor mPTP opening, which enables the study of various aspects of mPT in either isolated mitochondria or intact cells. The most popular mPT monitoring method using isolated mitochondria is based on measuring an increase in mitochondrial matrix volume with a mitochondrial swelling assay (usually detecting changes in the diffraction/ absorption of light measured at 540 nm)12,13. mPTP opening can also be measured indirectly as, for example, the dissipation of mitochondrial membrane potential ( ψm)8,14,15, elevated mitochondrial oxygen consumption (increased respiration)16, and the capture of radioactive compounds (such as C14-sucrose) in the mitochondrial matrix17 in response to activators of mPTP opening. Another extensively used procedure is the so-called calcium-retention capacity method18. This technique is based on the ability of mitochondria to accumulate Ca2+ in the matrix ([Ca2+]m) before triggering mPT. To exclude mPTP-independent factors, a specific amount of Ca2+, mitochondrial substrate, Pi, and adenine nucleotides has to be provided. This again implies the use of isolated mitochondria or (at least) permeabilized cells19,20 measured by spectrofluorometry. In all cases, it is necessary to confirm that the observed changes in the aforementioned measurement parameters are directly caused by mPTP opening. For this reason, as a gold standard to confirm mPT, cyclosporine A (CsA) is used to inhibit mPTP opening. CsA inhibition provides the most convincing evidence for mPT occurrence, as described in the literature. mPTP opening can also be monitored in vivo using radioactive deoxyglucose21. However, for estimation of the entrance of this compound into the mitochondrial matrix, isolation of mitochondria or at least plasma membrane permeabilization should be performed to eliminate the accumulation of radioactivity in the cytosol. This method has been used successfully to monitor mPTP opening during ischemia/reperfusion of the heart22.

有许多方法可用于监测mPT的开放程度，从而可以研究分离的线粒体或完整细胞中mPT的各个方面。使用分离的线粒体的最流行的mPT监测方法是基于用线粒体肿胀分析法测量线粒体基质体积的增加(通常检测在540 nm测量的光的衍射/吸收的变化)12，13。mPTP开放也可以间接测量为，例如，线粒体膜电位(ψm)8，14，15的消散，线粒体耗氧量升高(呼吸增加)16，以及响应于mPTP开放的激活剂在线粒体基质17中捕获放射性化合物(如C14-蔗糖)。另一个广泛使用的程序是所谓的calcium-retention capacity method18。该技术基于线粒体在触发mPT前在基质([Ca2+]m)中积累Ca2+的能力。为了排除与mPTP无关的因素，必须提供特定量的Ca2+、线粒体底物、Pi和腺嘌呤核苷酸。这再次意味着使用通过荧光光谱法测量的分离的线粒体或(至少)透化的细胞19，20。在任何情况下，都有必要确认上述测量参数中观察到的变化是由mPTP打开直接引起的。因此，作为确认mPT的金标准，环孢菌素A (CsA)用于抑制mPTP开放。如文献所述，CsA抑制为mPT的发生提供了最有说服力的证据。还可以使用放射性脱氧葡萄糖21在体内监测mPTP开放。但是，为了估计该化合物进入线粒体基质的情况，应进行线粒体分离或至少进行质膜透化，以消除细胞溶质中放射性的累积。该方法已成功用于监测心脏缺血/再灌注期间的mPTP开放22。

As described in detail in the present paper, assays of mPTP opening can be performed in living cells. These techniques avoid many of the artifacts (nonphysiological conditions) that accompany experiments using isolated mitochondria, and they offer the advantage of increased physiological relevance.

如本文详细描述的，可在活细胞中进行mPTP开放度测定。这些技术避免了许多伴随使用分离的线粒体的实验的伪影(非生理条件)，并且它们提供了增加生理相关性的优点。

Overview of the protocol

mPT is a fascinating but quite obscure phenomenon that requires careful investigation in living cells. For this reason, we describe three different methods that can be used to monitor mPTP activity: the Co2+ calcein assay, mitochondrial membrane depolarization, and the swelling technique.

mPT是一种令人着迷但相当模糊的现象，需要在活细胞中仔细研究。因此，我们描述了三种不同的方法来监测mPTP的活性:Co2+钙黄素测定法、线粒体膜去极化法和肿胀法。

The Co2+ calcein assay is a direct and efficient method for measuring mPTP opening in living cells that has been in use since the 1990s. This method can be used in a wide range of cell types and under many pathological conditions related to the mitochondria8,23 26. Cells are loaded with calcein dye (excitation/emmission: 494/517 nm), which can passively diffuse into the cells and collect in cytosolic compartments including the mitochondria. Once the dye is inside cells, esterases cleave the acetoxymethyl esters, trapping the dye in intracellular compartments. At this point, the living cells become fluorescently labeled, with fluorescence spread throughout all subcellular compartments. The utility of the method originates from the ability of cobalt to quench calcein fluorescence in the cytosol but not in mitochondria. Thus, in the case of healthy cells, calcein is able to reveal the mitochondrial matrix network with good localization (Fig. 1). Opening of the mPTP leads to both the exit of calcein from the mitochondrial matrix and entry of Co2+ into the mitochondrial matrix, resulting in quenching of calcein stored in the mitochondria. This event manifests as a reduction of calcein fluorescence intensity and is easily measurable by fluorescence microscopy27.

Co2+钙黄绿素分析法是一种直接有效的测量活细胞内mPTP开放度的方法，自20世纪90年代开始使用。该方法可用于多种细胞类型和许多与线粒体相关的病理条件下8，23 26。细胞负载有钙黄绿素染料(激发/发射:494/517 nm)，钙黄绿素染料可被动扩散到细胞中并聚集在包括线粒体在内的胞质区室中。一旦染料进入细胞内，酯酶会裂解乙酰氧基甲基酯，将染料捕获在细胞内的隔室中。此时，活细胞被荧光标记，荧光扩散到所有亚细胞区室。该方法的实用性源于钴在胞质溶胶中而非在线粒体中淬灭钙黄绿素荧光的能力。因此，在健康细胞的情况下，钙黄绿素能够以良好的定位揭示线粒体基质网络(图1)。mPTP的打开导致钙黄绿素从线粒体基质中排出，而Co2+进入线粒体基质，导致储存在线粒体中的钙黄绿素淬灭。该事件表现为钙黄绿素荧光强度降低，可通过荧光显微镜轻松测量27。

As mPTP opening leads to the loss of the proton gradient across membranes, the second assay measures this gradient using the Nernstian dye tetramethylrhodamine methyl ester (TMRM). TMRM is a cell-permeable, cationic, red-orange fluorescent dye. TMRM freely passes through cellular and mitochondrial membranes and, because of the large electrochemical H+ gradient across the mitochondrial inner membrane, accumulates in the mitochondrial matrix according to ψm. Opening of mPTP causes an efflux of TMRM from the mitochondria that manifests as a reduction of fluorescence intensity that is easily measurable by fluorescence microscopy (Fig. 2).

由于mPTP的打开会导致跨膜质子梯度的损失，第二种分析使用能斯特染料四甲基罗丹明甲酯(TMRM)测量该梯度。TMRM是一种细胞渗透，阳离子，红橙色荧光染料。TMRM自由穿过细胞膜和线粒体膜，并且由于穿过线粒体内膜的大电化学H+梯度，根据ψm在线粒体基质中积累。mPTP的打开导致TMRM从线粒体外排，表现为荧光强度的降低，这可通过荧光显微镜容易地测量(图2)。

The final assay to verify mPTP activity is the measurement of mitochondrial network integrity. Osmotic shock induced by mPTP opening allows for the uptake of H2O into the mitochondrial matrix and concomitant swelling of the inner mitochondrial membrane. This swelling causes mitochondria matrix expansion, which results in rupture of the outer mitochondrial membrane with loss of mitochondrial network integrity28 30. Overall in vivo mitochondrial swelling appears as a transformation of the mitochondrial network to a disorganized group of sphere-shaped structures. This can be quantified by counting the amount of objects composing the mitochondrial network when mitochondria are stained with specific fluorescent probes8,31 33 (Fig. 3).

验证mPTP活性的最终分析是测量线粒体网络的完整性。mPTP开放诱导的渗透休克允许H2O摄取到线粒体基质中，并伴随线粒体内膜的肿胀。这种肿胀会导致线粒体基质膨胀，从而导致线粒体外膜破裂，线粒体网络完整性丧失28 30。总体而言，体内线粒体肿胀表现为线粒体网络转化为一组无组织的球形结构。这可以通过在用特异性荧光探针8，31 33对线粒体进行染色时计数构成线粒体网络的对象的量来定量(图3)。

The procedure can be divided into four main and distinct sections. In the first section, seeded cells are stained to properly label mitochondria depending on the assay of interest (e.g., Co2+ calcein quenching, mitochondrial depolarization, or swelling). In the second step, it is essential to set up the imaging protocol to perform basal acquisitions that are necessary before treatment with chemical compounds to modulate inhibition or induction of the mPTP. Third, challenging the mPTP opening is the aim of the protocol, so a Ca2+- or reactive oxygen species (ROS)-dependent stimulus is added to the ongoing experiment. Finally, image processing and data analysis are performed to obtain a numeric index that describes mPTP activity and can be used for statistical data analysis.

该程序可分为四个主要和不同的部分。在第一部分中，对接种细胞进行染色，以根据目的分析(例如，Co2+钙黄绿素猝灭、线粒体去极化或肿胀)正确标记线粒体。在第二步中，必须建立成像方案，以进行基础采集，这在用化合物治疗以调节mPTP的抑制或诱导之前是必要的。第三，挑战mPTP开放是本方案的目的，因此在正在进行的实验中加入了Ca2+或活性氧(ROS)依赖性刺激。最后，进行图像处理和数据分析，以获得描述mPTP活动的数字索引，并可用于统计数据分析。

Potential applications of the protocol

The different methods described in this protocol can be used to measure mPTP opening in a wide range of intact cells. The use of the described fluorescent dyes, a controlled temperature, and the experimental setup allows for mitochondria to be present in their physiological environment. Under these conditions, the mPTP is regulated by the relevant mix of endogenous inhibitors and mPT activators in a manner that corresponds to in vivo conditions.

本方案中描述的不同方法可用于测量大范围完整细胞中的mPTP开放度。所述荧光染料的使用、受控温度和实验设置允许线粒体存在于它们的生理环境中。在这些条件下，mPTP受内源性抑制剂和mPT激活剂的相关混合物调节，调节方式与体内条件相对应。

Two major advantages are obvious when considering the use of intact cells compared with isolated mitochondria or permeabilized cells. First, the system is much more physiological. Given that the mPTP interfaces with different signaling pathways, these protocols allow investigations of signaling pathways afferent to the mPTP, putative mPTP components or regulators, or novel stimulators or inhibitors of the mPT. This could be easily achieved by exposure to medium enriched with compounds of interest, or by the use of molecular biology tools such as siRNA, shRNA, cDNA overexpression of wild-type or mutant proteins, Cas9 knockout, and mutant knock-in, among many other approaches. Second, live-cell imaging requires far fewer cells than mitochondrial isolation procedures or spectrofluorimetric techniques, which extends the field of application to cells that are not easily cultured.

当考虑使用完整细胞与分离的线粒体或透化细胞相比时，有两个主要优势是明显的。首先，这个系统更具生理性。鉴于mPTP与不同的信号通路相互作用，这些方案允许研究传入mPTP的信号通路、假定的mPTP组分或调节剂，或mPT的新型刺激因子或抑制剂。这可以很容易地通过暴露于富含目的化合物的培养基来实现，或者通过使用分子生物学工具，例如siRNA、shRNA、野生型或突变蛋白的cDNA过表达、Cas9敲除和突变敲除以及许多其他方法。其次，活细胞成像比线粒体分离程序或荧光光谱技术需要的细胞少得多，这将应用领域扩大到了不容易培养的细胞。

In our laboratory, this protocol has been successfully tested in HeLa cells, PC3 cells, HEK293T cells, SH-SY5Y cells, myoblasts, mouse embryonic fibroblasts, human adult fibroblasts, epithelial cells, HL-1 cells, rat neonatal cardiomyocytes, and CHO cells. Finally, these protocols are not time-consuming, the microscope setup is easy, and acquisition is fast. All methods apply different  techniques to achieve the same purpose, so that a positive (or negative) result that is confirmed by all methods provides a high degree of statistical power and reproducibility

 在我们的实验室中，已在HeLa细胞、PC3细胞、HEK293T细胞、SH-SY5Y细胞、成肌细胞、小鼠胚胎成纤维细胞、人成人成纤维细胞、上皮细胞、HL-1细胞、大鼠新生心肌细胞和CHO细胞中成功检测了该方案。最后，这些协议不耗时，显微镜设置简单，采集快速。所有方法都采用不同的技术来实现相同的目的，因此所有方法确认的阳性(或阴性)结果都具有高度的统计能力和再现性

Limitations of the protocol

该方案在悬浮培养的细胞或完整组织中不可行。这是因为在前一种情况下难以聚焦稳定的样本，而在后一种情况下组织切片的厚度或退化。此外，即使完整的细胞最适合用于检测这些方法，mPTP也不能被上述所有激活剂和mPT抑制剂直接利用。此外，在细胞质和线粒体基质中存在腺嘌呤核苷酸和Ca2+以及其他调节剂的单独池，也应被视为影响mPT的一个因素。或者，使用分离的线粒体研究mPTP功能可简化模型，并可研究mPT打开/关闭时的几种血浆膜透性化合物，还可通过调节例如氧化还原状态和不同离子的浓度来直接控制实验环境。

Experimental design

Analysis of mPTP activity in living cells requires comparison of the results among the three imaging techniques described in this protocol. Each assay challenges mPTP in a specific manner to verify a particular aspect of its activity, and global interpretation and integration of the data are necessary in order for the results of the experiments to be fully understood. The protocol is divided into the following main sections: sample preparation, fluorescence measurements, and analysis.

活细胞mPTP活性的分析需要比较本协议中描述的三种成像技术的结果。每一种分析都以特定的方式对mPTP进行挑战，以验证其活性的特定方面，为了充分理解实验结果，全球解释和数据整合是必要的。该方案分为以下主要部分:样品制备、荧光测量和分析。

Sample preparation.

The first phase is relatively flexible, and it allows researchers to optimize the protocol to their own experimental situations with respect to the following specifications. Concerning cell seeding, it is necessary that cells reach 50-70% confluence as a monolayer on the day of the experiment. Indeed, excessive cell density can interfere with stimulus delivery and weaken mPTP activity, leading to misunderstanding of the phenomena. Moreover, the choice of a suitable imaging coverslip for individual imaging setups and cell lines is important; for instance, glass coverslips with a suitable coating should be chosen on the basis of the cell adhesion.

第一阶段相对灵活，它允许研究人员根据他们自己的实验情况，根据以下规范优化方案。关于细胞接种，有必要在实验当天使细胞作为单层达到50-70%汇合。实际上，过度的细胞密度会干扰刺激传递并削弱mPTP活性，从而导致对现象的误解。此外，为各个成像设置和细胞系选择合适的成像盖玻片也很重要；例如，具有合适涂层的玻璃盖玻片应根据细胞粘附性来选择。

Cell staining and mitochondrial-labeling optimization should be specific for each of the three methods. We have found that problems may occur when calcein cobalt formation is incomplete, leaving a large amount of cytoplasmic calcein visible (see TROUBLESHOOTING). This should be verified by microscopic examination of the coverslip before the acquisition procedure is started. A mitochondrial counterstain may be particularly helpful, and in this procedure we suggest the use of Mitotracker Red. The mitochondrial accumulation of Mitotracker is dependent on ψm, and it can be partially quenched by calcein; indeed, this counterstaining is not intended for quantitative measurement and should not be included in the time-lapse acquisition.

细胞染色和线粒体标记优化应针对三种方法中的每一种。我们发现，当钙黄绿素钴形成不完全，留下大量胞质钙黄绿素可见时，可能会出现问题(请参阅故障诊断与排除)。在开始采集程序之前，应通过对盖玻片进行显微镜检查来验证这一点。线粒体复染可能特别有帮助，在该手术中，我们建议使用Mitotracker Red 。Mitotracker Red的线粒体积累依赖于ψm，并且可以被钙黄绿素部分淬灭；事实上，这种反染色不是用于定量测量的，也不应该包括在延时采集中。

For potentiometric measurements, TMRM is suggested, as it is probably one of the most common potentiometric dyes used. Nevertheless, alternative staining procedures that can lead to the same conclusion are described in Box 1.

对于电位测量，建议使用TMRM，因为它可能是最常用的电位测量染料之一。然而，可以得出相同结论的其他染色方法见框1。

Finally, the use of fluorescent reporters targeted to the mitochondrial matrix is strongly recommended for morphometric measurements because of their selective localization and resistance to photobleaching. The present procedures describe the use of mitochondrial GFP (mtGFP), but examples of alternatives that provide the same information are indicated in Table 1.

最后，强烈建议使用针对线粒体基质的荧光报告器进行形态测量，因为它们具有选择性定位和抗光漂白。本程序描述了线粒体GFP (mtGFP)的使用，但表1列出了提供相同信息的替代例子。

Fluorescence measurement.

Imaging setup is the most critical step of the procedure; this protocol describes the most common problems that may occur at this step. Particular attention should be paid to the exposure time and light intensity to avoid artifacts due to, for instance, photobleaching of the fluorescent probe. A correct setup allows the user to obtain optimal basal measurements that are fundamental to later challenges to mPTP opening. A variety of different mPT-inducing stimuli can be used; here we describe the use of two stimuli, [Ca2+]m (by means of the ionophore ionomycin) and ROS (by means of H2O2), that exert their effects in the range of seconds to tens of minutes, respectively. Nevertheless, alternative stimuli can be chosen depending on the experimental goals of the researcher; a list of potential alternatives is reported in Table 2. The time of sample acquisition varies depending on the stability of the reagents used, especially the probes. TMRM and mitochondrial fluorescent proteins are quite stable and can be monitored for up to 48 72 h. Conversely, calcein is somewhat rapidly extruded by the cell. In our hands, calcein signals display stable intensity for up to 46 60 min; indeed, for longer recording times, the user should set up the best conditions for the experimental situation. We also hasten to add that ionophores that can alter the plasma membrane potential34 can lead to mPT-independent TMRM re-distribution. Even if the combined use of a dye for plasma membrane potential allows for the correction of this artifact, the induced mitochondrial Ca2+ uptake generates an mPTP-independent mitochondrial depolarization35 that can produce experimental artifacts. Thus, even though several studies have successfully combined potentiometric dyes with ionophores to challenge mPTP opening, we recommend following this procedure, carefully evaluating the results, and considering different challenge methods (Table 2).

成像设置是该程序中最关键的步骤；此协议描述了在此步骤中可能发生的最常见问题。应特别注意暴露时间和光强，以避免因荧光探针光漂白等原因造成的伪影。正确的设置使用户能够获得最佳的基础测量值，这对以后打开mPTP是至关重要的。可以使用多种不同的mPT诱导刺激；这里我们描述了两种刺激的使用，[Ca2+]m(通过离子载体离子霉素)和ROS(通过H2O2)，它们分别在数秒至数十分钟的范围内发挥作用。然而，可以根据研究者的实验目标选择替代刺激；表2中报告了一份潜在替代品清单。样本采集时间取决于所用试剂的稳定性，尤其是探针的稳定性。TMRM和线粒体荧光蛋白非常稳定，可监测长达48-72小时。相反，钙黄绿素在一定程度上被细胞快速挤出。在我们手中，钙黄绿素信号显示稳定的强度长达46-60分钟；实际上，对于更长的记录时间，用户应该为实验情况设置最佳条件。我们还必须补充一点，可以改变质膜电势的离子载体34可以导致mPT独立的TMRM再分布。即使联合使用一种用于质膜电位的染料可以校正这种伪影，诱导的线粒体Ca2+摄取也会产生一种与mPTP无关的线粒体去极化35，从而产生实验性伪影。因此，尽管已有多项研究成功地将电位染料与离子载体相结合来挑战mPTP开放，但我们建议遵循此程序，仔细评估结果，并考虑不同的挑战方法(表2)。

Induction and inhibition of the mPTP.

Modulators of the mammalian mPTP can be divided into two classes: activators and inhibitors of mPT. mPT can be stimulated by several conditions and chemical compounds, such as accumulation of mitochondrial Ca2+ (ref. 36), ROS (superoxide) and pro-oxidative agents37,38, oxidized thiols39, Pi40,41, long-chain free fatty acids42, atractyloside43mastoparan44, and 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide (PK11195)45. In turn, mPT can be inhibited by high ψm; CsA; sanglifehrin A; bongkrekic acid; antamanide; 5-isothiocyanato-2-[2-(4-isothiocyanato-2- sulfophenyl)ethenyl]benzene-1-sulfonic acid; adenine nucleotides (ATP and ADP); creatine; cyclocreatine; glucose; König s polyanion; NADH; NADPH; UTP; ubiquinone; decylubiquinone; antioxidants; calcium chelators; and divalent cations such as Ba2+, Mg2+, Mn2+, and Sr2+. However, the conditions and compounds listed above can have no, or even opposing, effects on eukaryotic cells of different taxa (for further details, see ref. 46).

哺乳动物mPTP的调节剂可分为两类:mPT的激活剂和抑制剂。mPT可由多种条件和化合物刺激，如线粒体Ca2+积聚(参考。36)、ROS(超氧化物)和促氧化剂37、38、氧化硫醇39、Pi40、41、长链游离脂肪酸42、苍术酮43、天冬氨酸44和1-(2-氯苯基)-N-甲基-N-(1-甲基丙基)-3-异喹啉-甲酰胺(PK11195)45。反过来，高ψm可以抑制mPTCsA sanglifehrin Abongkrekic酸；antamanide5-异硫氰酸酯-2-[2-(4-异硫氰酸酯-2-磺基苯基)乙烯基]苯-1-磺酸；腺嘌呤核苷酸(ATP和ADP)；肌酸；环肌酸；葡萄糖；柯尼希的多妻制；NADHNADPHUTP；泛醌；去酰基泛醌；抗氧化剂；钙螯合剂；和二价阳离子如Ba2+、Mg2+、Mn2+和Sr2+。然而，上述条件和化合物可能对不同分类群的真核细胞没有影响，甚至相反(更多详情，见参考文献。46).

Data analysis. In this section, guidelines for image and data processing are comprehensively described to allow researchers who are not familiar with microscopy to perform the experiment. Specialists can optimize the analyses according to their own needs, but we recommend meticulously following the critical steps of the procedure.

数据分析。在本节中，图像和数据处理的指导方针被全面描述，以允许不熟悉显微镜的研究人员执行实验。专家可以根据自己的需要优化分析，但我们建议仔细遵循程序的关键步骤。

Experimental controls. As described before, CsA is a well-known inhibitor of mPT, and its activity is reported to prevent mPTP opening. Hence, we suggest including CsA treatment as a negative control in each experiment.

实验控制。如前所述，CsA是一种众所周知的mPT抑制剂，据报道其活性可以阻止mPTP的开放。因此，我们建议在每个实验中纳入CsA处理作为阴性对照。

Figure 1 | Representative images and kinetics of HeLa cells stained using the Co2+ calcein technique. Challenging cells with the ionophore ionomycin induced mPTP opening and quenching of the calcein signal. This event was inhibited by pretreatment with the mPTP-desensitizing agent CsA.

图1 |使用Co2+钙黄绿素技术染色的HeLa细胞的代表性图像和动力学。用离子载体离子霉素攻击细胞诱导了mPTP开放和钙黄绿素信号淬灭。通过使用mPTP脱敏剂CsA进行预处理，该事件得到抑制。

Figure 2 | Representative images and kinetics of HeLa cells stained with TMRM (Fire LUT was applied). Challenging the cells with the pro-oxidant H 2O2 induced mPTP opening, mitochondrial depolarization, and reduced TMRM signal intensity. This was inhibited by pretreatment with the mPTP-desensitizing agent CsA. Scale bars, 50 µm.

图2 TMRM染色HeLa细胞|代表图像和动力学(应用Fire LUT)。用促氧化剂h2o2刺激细胞，可诱导mPTP打开，线粒体去极化，并降低TMRM信号强度。mPTP脱敏剂CsA预处理可抑制这一现象。比例尺，50µm。

Figure 3 | Representative images and kinetics of HeLa cells expressing mitochondrially targeted GFP. Challenging the cells with the ionophore ionomycin induced mPTP opening, mitochondrial swelling, and mitochondrial network fragmentation, as represented by the increase in object count. This was inhibited by pretreatment with the mPTP-desensitizing agent CsA.

图3表达线粒体靶向GFP的HeLa细胞|代表图像和动力学。用离子载体离子霉素刺激细胞可诱导mPTP开放、线粒体肿胀和线粒体网络碎裂，如物体计数增加所示。mPTP脱敏剂CsA预处理可抑制这一现象。

Box 1 | Alternative mitochondrial membrane potential probes可选择的线粒体膜电位探针

In addition to TMRM, several fluorescent lipophilic cationic dyes, such as tetramethylrhodamine ethyl (TMRE) ester, Rhodamine 123 (RH-123), 3,3 -dihexyloxacarbocyanine iodide (DiOC6(3)), and JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide), are able to directly measure the mitochondrial membrane potential. As positively charged molecules, these dyes accumulate within mitochondria in inverse proportion to ψm according to the Nernst equation.

Different probes can be used for mPTP-induced mitochondrial depolarization; here, a brief description of each probe is provided, along with the modifications that researchers should make to the protocol.

TMRE is a cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria as TMRM. TMRM and TMRE are both quickly equilibrating dyes, but TMRM inhibits the electron-transport chain less, and it is recommended for this reason. TMRE application does not require modification of the protocol63.

RH-123 is lipophilic in nature, which allows it to diffuse through the mitochondrial membrane in response to potential and concentration gradients. Mitochondrial energization induces the quenching of RH-123 fluorescence, and the rate of fluorescence decay is proportional to the mitochondrial membrane potential. The application of RH-123 is suggested for short-time-scale (min) studies to monitor rapid step changes in ψm (ref. 63).

DiOC6(3) is a cell-permeant, green-fluorescent, lipophilic dye that, when used at low concentrations, is selective for the mitochondria of live cells. At higher concentrations, the dye may be used to stain other internal membranes, such as the endoplasmic reticulum. However, the use of DiOC6(3) is not recommended because of its higher toxicity toward mitochondrial respiration64.

 JC-1 is used as an indicator of mitochondrial potential in a variety of cell types. JC-1 exists as either a green-fluorescent monomer at depolarizing membrane potentials or a red-fluorescent aggregate at hyperpolarizing membrane potentials. The ratio of red to green fluorescence depends only on the membrane potential and not on other factors such as mitochondrial size, shape, and density, which may influence single-component fluorescence signals. All these mitochondrial probes are differentially permeable according to their distinct molecular structure, and JC-1 is the least permeable. More specifically, whereas the monomer form of JC-1 has been reported to equilibrate on a time scale similar to that of TMRM/TMRE (~15 min), the aggregate form of the dye takes ~90 min to equilibrate65. For this reason, the use of TMRM is suggested for this particular protocol.

方框1 |备选线粒体膜电位探针可选择的线粒体膜电位探针

除TMRM外，几种荧光亲脂性阳离子染料如四甲基罗丹明乙酯(TMRE)酯、罗丹明123 (RH-123)、3，3-二己基碳菁碘(dioch 6(3))、JC-1 (5，5，6，6-四氯-1，1，3，3-四乙基苯并咪唑基碳菁碘)等，均可直接测定线粒体膜电位。作为带正电荷的分子，根据能斯特方程，这些染料在线粒体内的累积量与ψm成反比。

不同探针可用于mPTP诱导的线粒体去极化；在此，提供了每个探针的简要描述，以及研究人员应该对方案进行的修改。

TMRE是一种透过细胞的阳离子红橙色荧光染料，很容易被活性线粒体(如TMRM)螯合。TMRM和TMRE都是快速平衡染料，但TMRM较少抑制电子传输链，因此建议使用。TMRE应用程序不需要修改协议63。

RH-123本质上是亲脂性的，因此可根据电位和浓度梯度通过线粒体膜扩散。线粒体通电诱导RH-123荧光猝灭，荧光衰减速率与线粒体膜电位成正比。建议将RH-123用于短时间尺度(min)研究，以监测ψm的快速阶跃变化(参考。63).

DiOC6(3)是一种细胞渗透，绿色荧光，亲脂性染料，当在低浓度下使用时，对活细胞的线粒体是选择性的。在较高浓度下，该染料可用于染色其他内膜，如内质网。然而，不建议使用二辛基6(3)，因为其对线粒体呼吸的毒性较高64。

JC-1被用作多种细胞类型中线粒体电位的指标。JC-1在去极化膜电位下以绿色荧光单体存在，在超极化膜电位下以红色荧光聚集体存在。红色与绿色荧光的比值仅取决于膜电位，而不取决于其他可能影响单组分荧光信号的因素，如线粒体大小、形状和密度。所有这些线粒体探针根据其独特的分子结构具有不同的通透性，其中JC-1的通透性最低。更具体地说，据报告，单体形式的JC-1在与TMRM/TMRE类似的时间尺度上达到平衡(~15分钟)，而染料的聚集形式需要~90分钟才能达到平衡65。因此，建议在此特定方案中使用TMRM。

additional reagents

· TMRE (Thermo Fisher Scientific, cat. no. T-669)

· RH-123 (Thermo Fisher Scientific, cat. no. R-302)

· DiOC6(3) (Thermo Fisher Scientific, cat. no. D-273)

· JC-1 dye (mitochondrial membrane potential probe; Thermo Fisher Scientific, cat. no. T-3168)

additional procedures

For the use of RH-123 in place of TMRM, the following step should be performed instead of that in the original PROCEDURE:

Step 2B(ii) Cell staining: add RH-123 working solution (10 M diluted in modified KRB) and incubate the cells for 20 min at 37 C in a 5% CO2 atmosphere. Anticipated results: using RH-123 to probe mPTP opening results in increased fluorescence, so pay particular attention to the interpretation of the data;

the slope values are positive and increase in proportion to mPTP opening.

For the use of DiOC6 in place of TMRM, the following step should be performed instead of that in the original PROCEDURE:

Step 2B(ii) Cell staining: add DiOC6(3) working solution (<1 nM diluted in modified KRB) and incubate the cells for 20 min at 37 C in a 5% CO2 atmosphere.

Step 3B Imaging setup

Set the acquisition frequency as indicated for TMRM in table 3.

For the use of JC-1 in place of TMRM, the following step should be performed instead of that in the original PROCEDURE:

· Step 2B(ii)—Cell staining: add JC-1 working solution (5 µM diluted in modified KRB) and incubate the cells for 30 min at 37 °C in

a 5% CO2 atmosphere. Remove the cells from the incubator and rinse them twice with 1 ml of modified KRB to remove the unbound

dye; then, add 900 µl of modified KRB.

· Step 3B(iii)—Imaging setup and basal acquisition:

Step 4B(ii) Challenging mPTP opening: after H2O2 addition, acquire images for at least 40 min.

Step 5B(i) Image processing: open the two time-lapse images obtained (one for red and one for green fluorescence) in the Fiji software. Create a time-lapse ratio image using the process image calculator and by dividing the intensity values for the red images by those for the green images. Then, using this time-lapse ratio image, proceed with the remaining image processing steps in the protocol.

Step 5B(ii) Image processing: on the FITC channel, draw an ROI; using freehand selections, encircle each mitochondrion, excluding the nucleus, and draw an ROI of a background region in an empty corner of the field.

Step 5B(iii) Image processing: for FITC and TRITC channels, estimate a global threshold to restrict the analysis to the pixels displaying intensity values greater than the threshold value. crItIcal step Estimate the threshold on the final image of the time-lapse.

 Step 5B(iv) Image processing: for FITC and TRITC channels, use the multimeasure tool to calculate the mean gray values limited by the threshold for the selected ROI (including the background ROI) in each time-lapse image.

Step 5B(vii) Image processing: normalize the values. Generate a new column representing the Ratio value; for each timepoint, divide the TRITC intensity by the FITC intensity. Then divide each timepoint value by the initial value, considering the first value as 100%.

 Step 5B(viii) Image processing: using the normalized Ratio column, calculate the slope from minute 15 to minute 25, for instance, using the function SLOPE in Microsoft Office Excel.

附加程序

要使用RH-123代替TMRM，应执行以下步骤，而不是原程序中的步骤:

步骤2B(ii)细胞染色:加入RH-123工作溶液(在改良KRB中稀释10 M)，并在37°C、5% CO2气氛下培养细胞20 min。预期结果:使用RH-123探测mPTP开口会导致荧光增加，因此要特别注意数据的解释；

斜率值为正，且与mPTP开口成比例增加。

如果使用教区6代替TMRM，应执行以下步骤，而不是原程序中的步骤:

步骤2B(ii)细胞染色:加入dioch 6(3)工作溶液(< 1 nM，在改良KRB稀释)，并在37°C、5% CO2气氛下培养细胞20 min。

步进3B成像设置

按照表3中的TMRM指示设置采集频率。

要使用JC-1代替TMRM，应执行以下步骤，而不是原程序中的步骤:

步骤2B(ii)-细胞染色:加入JC-1工作溶液(在改良KRB中稀释5 M)，在37°C下将细胞培养30 min

5%的CO2气氛。从培养箱中取出细胞，并用1 ml的改良KRB溶液冲洗两次，以去除未结合的细胞

染色；然后，加入900升改良KRB。

步骤3B(iii)—成像设置和基础采集:

步骤4B(ii)挑战mPTP开口:加入H2O2后，获取图像至少40 min。

步骤5B(i)图像处理:在斐济软件中打开获得的两幅延时图像(一幅用于红色荧光，一幅用于绿色荧光)。使用过程图像计算器并通过将红色图像的强度值除以绿色图像的强度值来创建延时比率图像。然后，使用该延时比率图像，继续协议中剩余的图像处理步骤。

步骤5B(ii)图像处理:在FITC通道上绘制感兴趣区域；使用徒手选择，环绕每个线粒体，不包括细胞核，并在视野的一个空角落画一个背景区域的感兴趣区域。

步骤5B(iii)图像处理:对于FITC和TRITC通道，估计一个全局阈值，将分析限制在强度值大于阈值的像素上。关键步骤评估延时的最终图像的阈值。

步骤5B(iv)图像处理:对于FITC和TRITC通道，使用多测量工具计算每个时间推移图像中选定ROI(包括背景ROI)的阈值限制的平均灰度值。

步骤5B(vii)图像处理:对值进行归一化。生成表示比率值的新列；对于每个时间点，用TRITC强度除以FITC强度。然后将每个时间点值除以初始值，将第一个值视为100%。

步骤5B(viii)图像处理:使用归一化比率列，计算从第15分钟到第25分钟的斜率，例如，使用Microsoft Office Excel中的函数slope。

PROCEDURE Cell preparation o TIMING 1 h

1 Seed cells onto a circular imaging dish, and allow the cells to grow until they reach 50% confluence. The researcher must optimize the seeding density for the given cell line. After seeding cells, wait for at least 24 h Cell staining and mitochondrial labeling

2 The following section describes how to perform sample staining. Please proceed to option A for Co2+-calcein, to option B for the TMRM method, and to option C for the mitochondrial morphology assay

（A）In vivo measurement of mPTP-induced Co2+-calcein quenching O TIMING 20 min

（i ）Remove the cells from the incubator, and wash them once with 1 ml of modified KRB to remove residual cellular debris and medium

7 TROUBLESHOOTING

（ii）Add 1 ml of calcein loading solution, and incubate the cells for 15 min at 37C in a 5%CO2 atmosphere A CRITICAL STEP Calcein is sensitive to light. Avoid exposure to light by handling the staining solution in low-light conditions and by protecting the sample with aluminum foil during the incubation step TROUBLESHOOTING

（iii）Remove the cells from the incubator and wash them twice with 1 ml of modified kRB to remove the unbound dye； then, add 900 ul of modified KRB.

A CRITICAL STEP Washing must be performed carefully Avoid the use of automatic aspiration tools

（B） In vivo measurement of mPTP-induced mitochondrial depolarization o TIMING 40 min

（Remove cells from the incubator, and wash them once with 1 ml of modified KRB to remove residual cellular debris and medium

7 TROUBLESHOOTING

（ii）Add 900 ul of TMRM loading solution, and incubate the cells for 30 min at 37C in a 5%CO2 atmosphere A CRITICAL STEP TMRM is sensitive to light. Avoid exposure to light by handling the solution in low-light conditions and by protecting the sample with aluminum foil during the incubation step.

TROUBLESHOOTING

（C）Measurement of in vivo mPTP-induced mitochondrial swelling O TIMING 90 min

（ Once the cells have reached 40-60% confluence, transfect them with 1-2 ug of mitochondrially targeted GFP per coverslip（ for equivalent reporters, see Table 1）。 The amount used for transfection depends on the chosen transfection lethod（usually 1 ug/cm2 for liposome- or polyethylenimine-based transfection and 2 ug/cm2 for Ca2+ phosphate-based transfection）。 After transfection, wait for 36-48 h

A CRITICAL STEP Each researcher should choose the appropriate transfection reagent and optimize the transfection protocol for the particular cell line being used. Alternatively, a stable cell line can be created using tandard protocols

程序时间为1小时的细胞制备

1将细胞接种到圆形成像皿上，让细胞生长直至达到50%汇合。研究人员必须优化给定细胞系的接种密度。接种细胞后，等待至少24小时细胞染色和线粒体标记

2下一节描述如何进行样本染色。请转到选项A(Co2+-钙黄绿素)、选项B(TMRM法)和选项C(线粒体形态分析)

(A)在体测量mPTP诱导的Co2+-钙黄绿素猝灭O计时20分钟

(I)从培养箱中取出细胞，并用1 ml改良KRB液清洗一次，以去除残留的细胞碎片和培养基

7故障诊断

(ii)加入1 ml的钙黄绿素加载溶液，并在37C、5%CO2气氛下培养细胞15 min。关键步骤钙黄绿素对光敏感。通过在弱光条件下处理染色溶液并在培养步骤中使用铝箔保护样本，避免光暴露故障诊断与排除

(iii)从培养箱中取出细胞，并用1 ml改性kRB清洗两次，以去除未结合的染料；然后，加入900 ul的改良KRB。

关键步骤必须小心进行清洗。避免使用自动抽吸工具

(B)在体测定mPTP诱导的线粒体去极化时间:40 min

(从培养箱中取出细胞，并用1 ml改良KRB液清洗一次，以去除残留的细胞碎片和培养基

7故障诊断

(ii)加入900 ul TMRM加载溶液，并在37C、5%CO2气氛下培养细胞30 min。关键步骤TMRM对光敏感。通过在弱光条件下处理溶液并在培养步骤中用铝箔保护样本，避免暴露于光。

解决纷争

(C)测量体内mPTP诱导的线粒体肿胀O时间点90分钟

(一旦细胞达到40-60%汇合，用每片盖玻片1-2 ug的线粒体靶向GFP转染细胞(等效报告子，见表1)。转染所用的量取决于所选择的转染剂量(基于脂质体或聚乙烯亚胺的转染通常为1微克/平方厘米，基于Ca2+磷酸盐的转染通常为2微克/平方厘米)。转染后，等待36-48小时关键步骤每个研究人员应针对所用的特定细胞系选择合适的转染试剂并优化转染方案。或者，可以使用标准方案产生稳定的细胞系

Imaging setup and basal acquisition

3 The following section describes how to set up for image acquisition. Please proceed to option A for Co2+-calcein to option B for the TMRM method, and to option C for the mitochondrial morphology assay

（A）In vivo measurement of mPTP-induced Co2+-calcein quenching o TIMING 1-5 min

（i）Place the imaging coverslip inside the temperature-controlled（37oC） microscope stage A CRITICAL STEP Living cells are sensitive to high and low temperatures. Be sure to maintain the temperature constant at37°C

（ii） Start the Xcellence Olympus software, and select the following specifications, which provide optimal image acquisition under standard conditions

A CRITICAL STEP Avoid extended and intense excitation of the sample

（iii） Focus on the sample and acquire a snapshot of a field containing cells that display a well-localized FItC signal in the mitochondrial compartment A CRITICAL STEP Calcein localization must overlap with the Mito Tracker signaL. Be sure to validate this result before the experiment

7 TROUBLESHOOTING

（iv） Strictly draw the regions of interest（ROIs） near the mitochondria of the selected cells for analysis of kinetic trends A CRITICAL STEP One ROI should always be dedicated to the background

（v） Start image acquisition at the frequency indicated in Table 3， and perform basal recording for 1 min TROUBLESHOOTING

成像设置和基础采集

3下一节描述如何设置图像采集。请转到选项A(对于Co2+-钙黄绿素)，转到选项B(对于TMRM法)，转到选项C(对于线粒体形态分析)

(A)在体测量mPTP诱导的Co2+-钙黄绿素猝灭时间:1-5 min

(I)将成像盖玻片放在温控(37oC)显微镜载物台A关键步骤活细胞对高温和低温敏感。确保将温度保持在37°C

(ii)启动Xcellence Olympus软件，并选择以下规格，这些规格可在标准条件下提供最佳的图像采集

关键步骤避免对样品进行长时间的强烈激发

(iii)聚焦样本并获取含有细胞的视野快照，这些细胞在线粒体区室中显示出良好定位的FItC信号。关键步骤钙黄绿素定位必须与米托跟踪器信号重叠。一定要在实验前验证这个结果

7故障诊断

(iv)严格绘制所选细胞线粒体附近的感兴趣区域(ROI)用于动力学趋势分析关键步骤一ROI应始终用于背景

(v)以表3中所示的频率开始图像采集，并进行1 min的基础记录故障诊断与排除

（B）In vivo measurement of mPTP-induced mitochondrial depolarization O TIMING 1-5 min

（i）Remove cells from the incubator.

A CRITICAL STEP Do not wash away the tmRm to avoid dye re-distribution

（ii）Place the imaging dish inside the temperature-controlled（37oC）microscope stage A CRITICAL STEP Living cells are sensitive to high and low temperatures. Be sure to maintain the temperature constant at37°C.

（iii） Start Xcellence Olympus software, and select the following specifications, which provide optimal image acquisition under standard conditions

 CRITICAL STEP Avoid extended and intense excitation of the sample

（iv） Select suitable field:we recommend selecting a field that displays no more than 70%o confluence of cells and that contains an empty corner.

TROUBLESHOOTING

（v） Strictly draw the ROIs near the mitochondria of the selected cells for analysis of kinetic trends

（vi） Start image acquisition at the frequency indicated in Table 3， and perform basal recording for 5 min TROUBLESHOOTING

(B)在体测定mPTP诱导的线粒体去极化O时间点1-5分钟

(I)从培养箱中取出细胞。

关键步骤不要洗掉tmRm以避免染料再分布

(ii)将成像皿放在温控(37oC)显微镜载物台A关键步骤活细胞对高温和低温敏感。确保将温度保持在37°c。

(iii)启动Xcellence Olympus软件，并选择以下规格，这些规格可在标准条件下提供最佳图像采集

关键步骤避免对样品进行长时间的强烈激发

(iv)选择合适的字段:我们建议选择显示不超过70%细胞汇合的字段，并且该字段包含一个空角。

解决纷争

(ii)严格将ROI画在所选细胞的线粒体附近，用于动力学趋势分析

(vi)以表3所示的频率开始图像采集，并进行5分钟的基础记录故障诊断与排除

（C） Measurement of in vivo mPTP-induced mitochondrial swelling. TIMING 1-5 min

（REmove the cells from the incubator, and extensively wash the cells with modified KRB

（ii）Cover the cells with 900 ul of modified KRB

（iii） Place the imaging coverslip inside the temperature-controlled （37C）microscope stage.

A CRITICAL STEP Living cells are sensitive to high and low temperatures. Be sure to maintain the temperature constant at37°C

（iv） Start the Xcellence Olympus software, and select the following specifications, which provide optimal image acquisition under standard conditions：

A CRITICAL STEP Avoid extended and intense excitation of the sample

（V） Focus on the sample and identify a field in which the cells display a well-localized mitochondrially targeted GFP signal in the mitochondrial compartment A CRITICAL STEP Be sure to select the central z plane of the cell to enable acquisition of the entire cell volume

（vi） Start image acquisition at the frequency indicated in Table 3， and perform basal recording for 3 min.

(mPTP诱导的线粒体体内肿胀测量。计时1-5分钟

(从培养箱中取出细胞，并用改良的KRB法彻底清洗细胞

(ii)用900 ul的改良KRB溶液覆盖细胞

(iii)将成像盖玻片置于温控(37C)显微镜载物台内。

关键步骤活细胞对高温和低温敏感。确保将温度保持在37°C

(iv)启动Xcellence Olympus软件，选择以下在标准条件下提供最佳图像采集的规格:

关键步骤避免对样品进行长时间的强烈激发

(V)聚焦样本并确定一个视野，在该视野中，细胞在线粒体区室中显示出定位良好的线粒体靶向GFP信号。关键步骤确保选择细胞的中心z平面，以便采集整个细胞体积

(vi)以表3所示的频率开始图像采集，并进行3 min的基础记录。

Challenging mPTP opening

4 The following section describes how to stimulate mPt during image acquisition. Please proceed to option a for Coz+-calcein, to option B for the TMRM method, and to option C for the mitochondrial morphology assay

（A）In vivo measurement of mPTP-induced Coz+-calcein quenching o TIMING 10 min

（i Add 100 ul of 10 uM ionomycin solution and mix it carefully A CRITICAL STEP Add the solution without touching the coverslip to avoid shifting the selected field

（i1） Perform acquisition for 9 min to ensure measurement of a response to the stimulus.

TROUBLESHOOTING BIn vivo measurement of mPTP-induced mitochondrial depolarization o TIMInG 30 min

（i Add 100 ul of 5 mM H202 solution and mix it carefully A CRITICAL STEP Add the solution without touching the coverslip to avoid shifting the selected field

（ii）Perform acquisition for 20 min to ensure measurement of a response to the stimulus.

TROUBLESHOOTING

（ii1）Add 100 ul of 10 uM FCCP solution and mix it carefully A CRITICAL STEP Add the solution without touching the coverslip to avoid shifting the selected field

（iv） Perform acquisition for 5 min to ensure measurement of a response to the stimulus.

TROUBLESHOOTING

（C）Measurement of in vivo mPTP-induced mitochondrial swelling O TIMING 10 min

（i Add 100 ul of 10 uM ionomycin solution and mix carefully A CRITICAL STEP Add the solution without touching the coverslip to avoid shifting the selected field

（i1） Perform acquisition for 15 min to ensure measurement of a response to the stimulus TROUBLESHOOTING

挑战mPTP开放

下节介绍在图像采集过程中如何刺激mPt。请继续选择方案a中的Coz+-钙黄绿素，选择方案B中的TMRM法，以及选择方案C中的线粒体形态分析

(A)在体测量mPTP诱导的Coz+-钙黄绿素猝灭时间:10 min

(I)加入100 ul 10uM离子霉素溶液并小心混合A关键步骤在不接触盖玻片的情况下加入溶液，以避免移动选定的视野

(i1)进行9 min的采集，以确保测量对刺激的反应。

故障诊断与排除

(B)In mPTP诱导的线粒体去极化的体内测量o TiME 30min

(I)加入100 ul 5mM H202溶液，并小心混合A关键步骤在不接触盖片的情况下加入溶液，以避免移动选定区域

(ii)采集20 min，以确保测量对刺激的反应。

解决纷争

(ii1)加入100 ul 10uM FCCP溶液，并小心混合A关键步骤在不接触盖玻片的情况下加入溶液，以避免移动选定区域

(iv)进行5分钟的采集，以确保测量对刺激的反应。

解决纷争

(C)测量体内mPTP诱导的线粒体肿胀O时间点10分钟

(I)加入100 ul 10uM离子霉素溶液并小心混合A关键步骤在不接触盖玻片的情况下加入溶液，以避免移动选定的视野

(i1)执行采集15 min，以确保测量对刺激的响应故障诊断与排除

（A）In vivo measurement of mPTP-induced Co2+-calcein quenching O TIMING 10 min Export the results as a spreadsheet by clicking"Save As"on the kinetic graph

（ii）Open the exported files using spreadsheet software, and subtract the background trace from each ROI arranged n a column cAlculate the slope of the curves for the first minute after ionomycin stimulation, for instance, using the function

"SLOPE in Microsoft office Excel

（iv） Input the slope values in a single column, and proceed with statistical analysis.

A CRITICAL STEP SLope calculation for periods of >1 min may underestimate the final results A CRITICAL STEP See Supplementary Video 1 for a video tutorial on this section（step 5A）

（B）In vivo measurement of mPTP-induced mitochondrial depolarization o TIMING 10 min

（ Open time-Lapse image using the Fiji software.

（ii）Draw an ROI； using freehand selections, encircle each mitochondrion, excluding the nucleus, and draw an ROI of a background region in an empty comer of the field.

i）Estimate a global threshold to restrict the analysis to the pixels displaying intensity values greater than the threshold value A CRITICAL STEP Estimate the threshold on the final image of the time-lapse

（iv）Use the multimeasure tool to calculate the mean gray values limited by the threshold for the selected ROI

（including the background Ror） in each time-Lapse image.

（v） Export the data in a spreadsheet-compatible format （i.e. Microsoft Office Excel） and arrange the data for each ROI in a single column.

n） For every column, subtract the background value at the sampling time.

（vii）Normalize the values, considering the first value as 100%o

（viii） Calculate the slope from minute 15 to minute 25， for instance, using the function SLOPE in Microsoft Office Excel A CRITICAL STEP Every trace must be analyzed using the same parameters, especially in the time -Lapse slope calculation

（ix） Use the slope values to perform statistical analysis, and compare the results under different experimental conditions.

A CRITICAL STEP See Supplementary Video 2 for a video tutorial on this section（step 5B）

（C）Measurement of in vivo mPTP-induced mitochondrial swelling o TIMING 30 min Select the last acquired time-lapse image and apply 3D digital deconvolution. Ensure that Xr and Z calibration and the refraction index of the lens are properly set. Be sure to perform automated spherical aberration detection

（ii）Export the deconvolved time-lapse image as multiple TIFF or Image Cytometry Standard files Open Fiji software and load the generated files.

2 TROUBLESHOOTING

（iv） Split different time points using the tool 'Stack Splitter； set the number of substacks as the number of time points

（ Menu Image→ Stack→ Tools）

（v）Select time point 1 and open the 3d object counter tool（Menu analyze）。

n）Set the threshold to include all mitochondria and to exclude the background. Set the minimum object size to ten pixels to avoid noise contamination

（vii）Record the number of objects counted

（viii） Repeat this procedure for all time points.

（ix）Collect all object counts in a spreadsheet file, arranging the data by row or column A CRITICAL STEP See Supplementary Video 3 for a video tutorial on this section （step 5C）

7 TROUBLESHOOTING

(mPTP诱导的Co2+钙黄绿素猝灭O时间10 min的体内测量

(i)通过单击动力学图上的“另存为”将结果导出为电子表格

(ii)使用电子表格软件打开导出的文件，并从排列在一列中的每个ROI中减去背景轨迹计算离子霉素刺激后第一分钟的曲线斜率，例如，使用函数

Microsoft office Excel中的“斜率”

(iv)将斜率值单列输入，并进行统计分析。

关键步骤斜率计算时间> 1 min可能会低估最终结果关键步骤参见补充视频1了解本部分的视频教程(步骤5A)

(二)在体测定mPTP诱导的线粒体去极化o时间点10分钟

(使用Fiji软件打开延时图像。

(二)绘制ROI；使用徒手选择，环绕每个线粒体，不包括细胞核，并在视野的一个空角落画一个背景区域的感兴趣区域。

I)估计全局阈值，以将分析限制为显示强度值大于阈值的像素关键步骤估计延时的最终图像上的阈值

(iv)使用多测量工具计算选定ROI的受阈值限制的平均灰度值

(包括背景误差)在每张延时图像中。

(v)以电子表格兼容格式(即Microsoft Office Excel)导出数据，并将每个投资回报的数据排列在一列中。

n)对于每一列，减去采样时的背景值。

(vii)将数值标准化，将第一个数值视为100%o

(viii)计算从第15分钟到第25分钟的斜率，例如，使用Microsoft Office Excel中的函数Excel A CRITICAL STEP必须使用相同的参数分析每个轨迹，尤其是在时间推移斜率计算中

(ix)利用斜率值进行统计分析，比较不同实验条件下的结果。

关键步骤参见补充视频2了解本部分的视频教程(步骤5B)

(C)测量体内mPTP诱导的线粒体肿胀o时间点30 min

(i)选择最后一次采集的延时图像并应用3D数字反卷积。确保Xr和Z校准以及透镜的折射率设置正确。确保执行自动球面像差检测

(ii)将去卷积的延时图像导出为多个TIFF或Image Cytometry标准文件打开斐济软件并加载生成的文件。

2故障诊断

(iv)使用工具“堆栈拆分器”拆分不同的时间点；将子包的数量设置为时间点的数量

(菜单图像→堆栈→工具)

(v)选择时间点1并打开3d对象计数器工具(菜单分析).

n)设置阈值以包括所有线粒体并排除背景。将最小对象大小设置为十个像素，以避免噪声污染

(七)记录清点的物品数量

(viii)对所有时间点重复此程序。

(ix)收集电子表格文件中的所有对象计数，按行或列排列数据A关键步骤有关此部分的视频教程，请参见补充视频3(步骤5C)

7故障诊断

Experiment and reagent setup: ~1 week

Step 1, cell preparation: 1 h

Step 2, cell staining and mitochondrial labeling

Step 2A, in vivo measurement of mPTP-induced Co2+-calcein quenching: 20 min

Step 2B, in vivo measurement of mPTP-induced mitochondrial depolarization: 40 min

Step 2C, measurement of in vivo mPTP-induced mitochondrial swelling: 90 min

Step 3, imaging setup and basal acquisition

Step 3A, in vivo measurement of mPTP-induced Co2+–calcein quenching: 1–5 min

Step 3B, in vivo measurement of mPTP-induced mitochondrial depolarization: 1–5 min

Step 3C, measurement of in vivo mPTP-induced mitochondrial swelling: 1–5 min

Step 4, challenging mPTP opening

Step 4A, in vivo measurement of mPTP-induced Co2+–calcein quenching: 10 min

Step 4B, in vivo measurement of mPTP-induced mitochondrial depolarization: 30 min

Step 4C, measurement of in vivo mPTP-induced mitochondrial swelling: 10 min

Step 5, image processing and data analysis

Step 5A, in vivo measurement of mPTP-induced Co2+–calcein quenching: 10 min

Step 5B, in vivo measurement of mPTP-induced mitochondrial depolarization: 10 min

Step 5C, measurement of in vivo mPTP-induced mitochondrial swelling: 30 min

box 1, alternative mitochondrial membrane potential probes: 80–85 min

实验和试剂设置:~1周

步骤1，细胞制备:1小时

步骤2，细胞染色和线粒体标记

步骤2A，mPTP诱导的Co2+钙黄绿素淬灭的体内测量:20分钟

步骤2B，mPTP诱导的线粒体去极化的体内测量:40分钟

步骤2C，测量体内mPTP诱导的线粒体肿胀:90分钟

第3步，成像设置和基础采集

步骤3A，mPTP诱导的Co2+–钙黄绿素淬灭的体内测量:1–5分钟

步骤3B，mPTP诱导的线粒体去极化的体内测量:1-5分钟

步骤3C，测量体内mPTP诱导的线粒体肿胀:1-5分钟

第4步，挑战mPTP的开放

步骤4A，体内测定mPTP诱导的Co2+–钙黄绿素猝灭:10分钟

步骤4B，mPTP诱导的线粒体去极化的体内测定:30 min

步骤4C，测量体内mPTP诱导的线粒体肿胀:10分钟

第五步，图像处理和数据分析

步骤5A，mPTP诱导的Co2+–钙黄绿素淬灭的体内测量:10分钟

步骤5B，mPTP诱导的线粒体去极化的体内测定:10 min

步骤5C，体内mPTP诱导的线粒体肿胀测量:30 min

方框1，备选线粒体膜电位探针:80-85分钟

antIcIpateD results

During analysis of mPTP activity by the calcein Co2+ technique, basal measurements should produce a stable signal. If the signal does not seem stable or if calcein displays a localization pattern that is different from that of MitoTracker (or another counterstain, if used), please refer to Table 4 for Troubleshooting guidelines (supplementary Fig. 1). Stimulating cells with ionomycin facilitates the entry of excess Ca2+ into cells to trigger mPTP opening. This event causes Co2+ entry into the mitochondria, thereby quenching the calcein signal. Indeed, kinetic analysis should produce a slope that adequately reflects mPTP opening. The response to ionomycin can be partially blocked using CsA, a compound that has been reported to desensitize mPTP formation by binding to cyclophilin D50. Thus, under this experimental condition, calcein is not quenched after mPTP challenge, and the slope of the time-dependent calcein intensity should be nearly zero (Fig 1).

As oxidative stress is one of the best characterized homeostatic perturbations that promotes mPTP opening51,52, the mitochondria of cells exposed to the prototypic pro-oxidant H2O2 underwent rapid depolarization. As described in the PROCEDURE section, the cells were stained with TMRM, a cell-permeant, cationic, red-orange fluorescent dye that is sensitive to ψm. The time-lapse images were analyzed and converted to a plot as described above. In Figure 2, representative images of TMRM staining under basal, H2O2-stimulated, and residual polarization conditions and a representative trace are reported. TMRM fluorescence was stable for the first 5 min (basal conditions). Subsequently, challenge with 500 M H2O2 induced a gradual decrease in the TMRM signal due to mPTP-opening-dependent depolarization. The slope of the trace directly correlates to mPTP opening. The slope of each trace derived from single cells can be used to compare different experimental conditions (i.e., vehicle versus CsA treatment). At the end of the experiment, FCCP, a mitochondrial electron-chain-uncoupling agent, was added to induce complete mitochondrial membrane depolarization. The residual TMRM staining intensity is due to non-mitochondrial membrane potential for instance, cellular membrane potential which should be excluded from analysis.

 Traces obtained from the analysis are suitable indexes for interpreting when an experiment is not properly set up. supplementary Figure 1 shows two examples of the most common artifactual results; please also refer to the Troubleshooting guidelines.

Challenging cells with ionomycin leads to the deformation of mitochondrial filaments and transforms them into groups of spheroidal objects (Fig 3). The number of mitochondria obtained according to the instructions in this protocol is expected to markedly increase. This mitochondrial network rearrangement is inhibited by pretreatment with the mPTP-desensitizing agent CsA. If mitochondria sense some environmental stress (i.e., excess light illumination), then undesired fragmentation might be observed before the mPTP stimulation. In the case of unfortunate events such as that described, please refer to the Troubleshooting guidelines (supplementary Fig. 1).

预期结果

在用钙黄绿素Co2+技术分析mPTP活性期间，基础测量应产生稳定的信号。如果信号似乎不稳定，或者钙黄绿素显示的定位模式不同于MitoTracker(或另一种复染剂，如果使用)的定位模式，请参阅表4了解故障诊断指南(补充图1)。用离子霉素刺激细胞有助于过量Ca2+进入细胞，从而触发mPTP开放。该事件导致Co2+进入线粒体，从而淬灭钙黄绿素信号。事实上，动力学分析应产生一个斜率，足以反映mPTP的开放。使用CsA可以部分阻断对离子霉素的反应，据报道，CsA是一种通过与亲环素D50结合使mPTP形成脱敏的化合物。因此，在该实验条件下，钙黄绿素在mPTP激发后未被淬灭，且依赖于时间的钙黄绿素强度的斜率应接近于零(图1)。

由于氧化应激是促进mPTP开放的最具特征的稳态扰动之一51，52，暴露于原型前氧化剂H2O2的细胞的线粒体经历了快速去极化。如程序部分所述，使用TMRM(一种对ψm敏感的细胞通透性阳离子红橙色荧光染料)对细胞进行染色。分析了时间推移图像，并将其转换为上述图。在图2中，报告了基础、H2O2刺激和残留偏振条件下TMRM染色的代表性图像和代表性痕迹。TMRM荧光在前5分钟(基础条件)是稳定的。随后，用500 µM H2O2激发导致TMRM信号逐渐减弱，这是由于mPTP的开放依赖性去极化所致。轨迹的斜率与mPTP开口直接相关。来源于单细胞的每种微量元素的斜率可用于比较不同的实验条件(即赋形剂与CsA治疗)。实验结束时加入线粒体电子链解偶联剂FCCP，诱导线粒体膜完全去极化。残余TMRM染色强度是由于非线粒体膜电位所致，例如，应排除在分析之外的细胞膜电位。

当实验设置不当时，从分析中获得的痕迹是解释的合适指标。补充图1显示了最常见的人工结果的两个例子；请同时参阅故障诊断与排除指南。

用离子霉素攻击细胞导致mitochondrial filaments and transforms them into groups of spheroidal objects(图3)。根据本方案中的说明获得的线粒体数量预计将显著增加。这种线粒体网络重排受到mPTP脱敏剂CsA预处理的抑制。如果线粒体感觉到一些环境应激(即光照过量)，则可能在mPTP刺激前观察到不希望的碎片。如果发生上述不幸事件，请参阅故障诊断与排除指南(补充图1)。