image analysis of  hep-3b cells after drug treatment

定量 秀丽隐杆线 虫和肝癌细胞系中的线粒体自噬

Mitochondria are essential for all aerobic animals, including humans. They convert the chemical energy of biomolecules to adenosine triphosphate (ATP) via oxidative phosphorylation1, synthesize heme2, degrade fatty acids through &#946; oxidation3, regulate calcium4 and iron5 homeostasis, control cell death by apoptosis6, and generate reactive oxygen species (ROS), which play a vital role in redox homeostasis7. Two complementary and opposite processes maintain the integrity and proper function of the mitochondria: the synthesis of new mitochondrial components (biogenesis) and the selective removal of damaged ones through mitochondrial autophagy (i.e., mitophagy)8.
Several mitophagy pathways are mediated by enzymes, such as PINK1/Parkin, and receptors, including FUNDC1, FKBP8, and BNIP/NIX9,10. Notably, the selective degradation of mitochondrial components can occur independently of the autophagosome machinery (i.e., through mitochondrial-derived vesicles)11. However, the endpoints of the different selective mitophagy pathways are similar&#160;(i.e.,mitochondrial degradation by lysosomal enzymes)12,13. For this reason, various methods for identifying and measuring mitophagy rely on the colocalization of mitochondrial and lysosomal markers14,15,16,17 and decreased levels of mitochondrial proteins/mitochondrial DNA18.
Below is a concise description of the existing experimental methodologies for measuring mitophagy in animal cells using fluorescence microscopy, emphasizing the mitophagy endpoint phase.
Mitophagy biosensors 
Mitochondrial degradation occurs within the acidic environment of the lysosome19. Therefore, mitochondrial components, including proteins, experience a shift from a neutral to an acidic pH at the endpoint of the mitophagy process. This pattern underpins the mechanism of action of several mitophagy biosensors, including mito-Rosella18 and tandem mCherry-GFP-FIS114. These sensors contain a pH-sensitive green fluorescence protein (GFP) and a pH-insensitive red fluorescence protein (RFP). Therefore, at the endpoint of mitophagy, the green-to-red fluorescence ratio drops significantly due to the quenching of the GFP fluorophore. The major limitations of these sensors are (1) possible F&#246;rster resonance energy transfer (FRET) between the fluorophores; (2) the differential maturation rate of GFP and RFP; (3) dissociation between the GFP and RFP due to proteolytic cleavage of the polypeptide that connects them; (4) fluorescence-emission overlap; and (5) differential fluorophore brightness and quenching15,16.
A sensor that overcomes some of these limitations is the Keima mitochondrial sensor17. The mt-Keima sensor (derived from the coral protein Keima) displays a single emission peak (620 nm). However, its excitation peaks are pH-sensitive. As a result, there is a transition from a green excitation (440 nm) to a red one (586 nm) when shifting from a high pH to an acidic pH16,17. A more recent mitophagy sensor, Mito-SRAI, has advanced the field by allowing for measurements in fixed biological samples20. However, despite the many advantages of genetic sensors, such as the ability to express them in specific tissues/cells and target them to distinct mitochondrial compartments, they also have limitations. One limitation is that the genetic sensors need to be expressed in cells or animals, which can be time-consuming and resource-intensive.
Additionally, the expression of the sensors within mitochondria themselves may influence the mitochondrial function. For example, expressing mitochondrial GFP (mtGFP) in the C. elegans worm body wall muscles expands the mitochondrial network21. This phenotype depends on the function of the stress-activated transcription factor ATFS-1, which plays an essential role in the activation of unfolded protein response in mitochondria (UPRmt)21. Therefore, although genetically encoded mitochondria/mitophagy biosensors are extremely useful for monitoring mitochondria homeostasis in vivo, they may affect the very process they are designed to measure.
Mitochondria/lysosome-specific antibodies and dyes 
Another strategy for testing mitochondrial/lysosome colocalization is to use antibodies against mitochondrial/lysosomal proteins, such as the mitochondrial outer membrane protein TOM20 and lysosomal-associated membrane protein 1 (LAMP1)22. In most cases, secondary antibodies that are conjugated to a fluorophore are used to detect the fluorescence signal via microscopy. Another strategy is to combine genetic constructs with mitochondrial/lysosomal dyes, such as expressing a LAMP1::GFP fusion construct in cells while staining them with a red mitochondrial dye (e.g., Mitotracker Red)16. These methodologies, while effective, require specific antibodies and often involve working with fixed specimens or generating cells/transgenic animals expressing fluorescently labeled mitochondria/lysosomes.
Here, we outline the utilization of a commercial lysosome/mitochondria/nuclear staining kit for assessing the mitophagy-activating properties of synthetic diamine O,O (octane-1,8-diyl)bis(hydroxylamine), hereafter referred to as VL-85023, in C. elegans worms and the human cancer cell line Hep-3B (Figure 1). The staining kit contains a mixture of lysosomal/mitochondrial/nuclear-targeted dyes that specifically stain these organelles23. We previously used this kit to demonstrate the mitophagy activity of 1,8 diaminooctane (hereafter referred to as VL-004) in C. elegans23. Importantly, we validated the staining kit results with the mito-Rosella biosensor and qPCR measurements of the mitochondrial:nuclear DNA content23. This staining kit offers the following advantages. First, there is no need to generate transgenic animals or cells expressing a mitochondrial biosensor. Therefore, we can study unmodified wild-type animals or cells and, thus, save much time, money, and labor. Moreover, as mentioned, expressing mitochondrial biosensors can change the mitochondrial function. Second, the kit is cost-effective, easy to use, and fast. Third, although we demonstrate the method in C. elegans and human cells, it could be modified for other cell types and organisms.
With that said, like any method, the staining kit protocol has drawbacks. For example, the incubation of the worms with the reagent is carried out in the absence of food (we have seen that even dead bacteria significantly decrease the staining efficiency). Although the incubation time is relatively short, it is possible that even in this time frame, homeostatic responses may be altered, including mitophagy. In addition, the binding of the dyes to the ER/mitochondrial/nuclear proteins and other biomolecules may affect the activities of these organelles. Moreover, unlike mitophagy measurement with genetic sensors, we work with worms and cells that have undergone chemical fixation. Therefore, it is impossible to continue monitoring the same worms/cells at different times. Hence, we recommend combining different methodologies to validate the function of mitophagy in a particular physiological process. Below, we present new data demonstrating that VL-850 induces robust mitophagy in C. elegans worms and Hep-3B cells. Therefore, these data further support the hypothesis that VL-850 extends the lifespan of C. elegans and protects C. elegans from oxidative damage through the induction of healthy mitophagy. We have used the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), which is a potent mitophagy inducer24, as a positive control.

作者聚焦：使用细胞器特异性染料检测秀丽隐杆线虫和哺乳动物细胞中的线粒体自噬  

使用细胞器特异性染料检测 秀丽隐杆 线虫和哺乳动物细胞中的线粒体自噬

Degradation of damaged mitochondria by mitophagy is essential for the function of the mitochondrial network. Unfortunately, this process declines with age. We develop and use drugs that activate mitophagy, thereby promoting healthy aging to understand the function of mitophagy in aging and age-related diseases, including Alzheimer's.One of the biggest challenges is to measure the mitophagy process without affecting it. Recent studies have shown that GFP expression in the mitochondria of muscle cells induces stress that is MT UPR. Therefore, using dyes in non-transgenic animals or cells is essential and complements genetic technologies.Our protocol has three main advantages. First, there is no need to generate transgenic animals or cells expressing mitophagy biosensors. Second, no need for expensive infrastructure and expertise such as in case of electron microscopy.And third, the organelle dye cocktail is commercially available and the protocol is simple and fast. The new mitophagy-activating compound VL-850 induces robust mitophagy, and in this way protects from oxidative stress and promote lifespan. Now the big question is to determine the underlying mechanism of action.We explored the mechanism at multiple levels, from the cellular to the organismal using C.elegans and mammalian cells.

Watch this Scientific Journal Video about Image Analysis of  Hep-3B Cells after Drug Treatment at JoVE.com

Image Analysis of  Hep-3B Cells after Drug Treatment  

药物治疗后Hep-3B细胞的图像分析

对药物处理的Hep-3B细胞进行成像后，使用共定位插件打开图像J中的共聚焦图像。在图像J服务器中打开ND文件，然后在对话框中选择分割通道选项。处理绿色和红色图像。生成这些图像的副本以保持原始图像不变，方法是单击图像并选择重复或使用键盘快捷键 shift 以及 D.To 减少背景，生成另一个图像副本，减去滚动半径为 100 的背景，然后选择创建背景选项以生成具有给定图像背景的图像。然后，转到处理。单击图像计算器并从第二个重复图像中减去第一个重复的图像。利用生成的图像进行共定位分析。要使用共定位插件，请通过单击图像，选择类型，然后选择 8 位，将绿色和红色通道图像转换为 8 位。接下来，单击插件并选择共定位。要测量线粒体和溶酶体信号的共定位，请将比率设置为 75%，阈值红色通道设置为 80.0，阈值绿色通道设置为 50.0。将生成一个包含共定位点并将三个 8 位图像组合在 RGB 图像中的 8 位二进制图像。将这些图像转换为 8 位图像。要获取像元的感兴趣区域，请通过选择共定位点的图像来生成突出显示像元区域的图像。要选择整个像元区域，请选择生成的共定位点图像，然后单击图像，调整阈值并设置阈值以确保突出显示所有像元区域。要分析像元的区域，请选择分析，单击分析粒子，然后测量图像中从零到无穷大的所有粒子，这是分析粒子的默认设置。要分析共定位线粒体和溶酶体的区域，请选择共定位的 8 位图像。然后，选择“分析”。单击分析颗粒并测量 0.1625 平方微米和 4 平方微米之间的点和。将共定位线粒体和溶酶体的面积除以总细胞面积以测量共定位点。Hep-3B细胞的共聚焦图像显示线粒体和溶酶体的共定位。VL 850和FCCP诱导Hep-3B细胞中显着的线粒体自噬。

image-analysis-of-hep-3b-cells-after-drug-treatment

Research

JoVE Journal

Biology

Image Analysis of  Hep-3B Cells after Drug Treatment

91 次观看.  The Hebrew University of Jerusalem. 对药物处理的Hep-3B细胞进行成像后，使用共定位插件打开图像J中的共聚焦图像。在图像J服务器中打开ND文件，然后在对话框中选择分割通道选项。处理绿色和红色图像。生成这些图像的副本以保持原始图像不变，方法是单击图像并选择重复或使用键盘快捷键 shift 以及 D.To 减少背景，生成另一个图像副本，减去滚动半径为 100 的背景，然后选择创建背景选项以生成具有给...

视频: Image Analysis of  Hep-3B Cells after Drug Treatment  

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

科研

生物学

Protocol for Measuring Mitophagy in Caenorhabditis elegans

Protocol for Measuring Mitophagy in Caenorhabditis elegans  

To begin, take the synchronized young adult C.Elegans hermaphrodites in a micro centrifuge tube. Add 200 microliters of M9 buffer, containing Poloxamer 188, Pluronic F127, and two microliters of the staining kit reagent to the worm pellet. Cover the tubes with aluminum foil to protect the dyes from light.Let the mixture rotate at 20 RPM for one hour at room temperature. Then spin the worms at 500 G for one minute. After that, remove the staining solution without disturbing the worm pellet.Wash the worms with M9 buffer thrice and transfer them into a seeded NGM agar plate containing the appropriate treatment. Wash the worms off the plates into fresh micro centrifuge tubes with one milliliter of M9 buffer. Afterward, fix the worms with 1%formaldehyde on ice for 30 minutes.And wash the worms with one milliliter of M9 buffer thrice to remove any residual formaldehyde. After pelleting the worms, aspirate the maximum amount of supernatant, keeping the pellet intact in 10 microliters of M9.Next, to prepare 2.5%agarose, weigh 0.125 grams of agarose into a 10 milliliter borosilicate glass test tube. Add five milliliters of M9 buffer and dissolve the agarose by gently heating the tube with a bunsen burner.Transfer the melted agarose to a dry bath set at 75 degrees Celsius. Add 100 microliters of the melted agarose onto a deck glosser microscope cover glass using a one milliliter tip. Immediately, put another slide perpendicularly on the agarose drop forming a cross shape.After two minutes, gently separate the slides by pushing the upper cover glass leaving the agarose pad on the bottom cover slide. Transfer the worms onto the agarose pad with a Pasteur glass pipette. Use a wick made of a laboratory wipe to remove excess liquid.Then cover the worms with a smaller cover slip and apply transparent nail polish to the periphery to prevent evaporation. Place the slide in a dark box to protect it from light. Use a confocal microscope to image the worms within 24 hours at the appropriate wavelengths using a 60 times magnification lens.Next, open the imaging software and right click on the gray area. From the popup that appears, select acquisition. Ti2 full pad.ND acquisition, and lookup tables. Under Ti2 full pad, select 60 times and adjust the microscope fine focus knob to bring the worms into focus. Then select spinning disc and choose the 16 bit-no binning option.For each fluorescence filter, set the exposure time to 500 milliseconds and 20 milliseconds for bright field. Once these parameters are set, select run now and wait for the output image to be generated as an ND2 file.

测量秀丽隐杆线虫线粒体自噬的协议

Analyzing the Images of Caenorhabditis elegans after Drug Treatment

Analyzing the Images of Caenorhabditis elegans after Drug Treatment  

After imaging the drug-treated C.elegane, open the ND file in the image J server with the colocalization plugin and select the split images option in the dialogue box. Each ND file contains image planes taken at three wavelengths and visible light. Work with brightfield, green, and red images.Generate duplicates of these images to keep the original image untouched by clicking on image and selecting duplicate or using the keyboard shortcut shift plus D.Then reduce the background by generating another image duplicate as demonstrated earlier. Subtract the background with a rolling radius of 100 and select the create background option to generate an image with the given image's Background. Now go to process, click image calculator and subtract the first duplicated image from the second duplicated image.Use the resulting images for the colocalization analysis. To use the colocalization plugin, convert the green and red channel images to eight bit by clicking image, selecting type, followed by eight bit. Next, click on plugins and select colocalization.To measure the colocalization of mitochondria and lysosome signals, set the ratio to 75%the threshold red channel to 80.0, and the threshold green channel to 50.0. An eight bit binary image containing colocalized puncta and combining the three eight bit images in an RGB image is generated as output. Next, focus on the puncta in the head body wall muscle of the worms by manually selecting the area and creating a mask by clicking edit, selection, and create mask to select the region of interest.To select the particles in the region of interest, use the image calculator to select the colocalized eight bit and masked images. Utilize the operation and to select the puncta in the region of interest generating an image with puncta in the region of interest. Finally, to analyze the area of the colocalized mitochondria and lysosomes, select analyze, followed by analyze particles and measure the summation of the puncta between 0.1625 square micrometers and four square micrometers.The confocal images of the head body wall muscles of worms showed colocalization of mitochondria and lysosomes. VL-850 induced robust mitophagy in the worm's head body wall muscles, indicating a potent mitophagy inducer. The mitophagy potency of VL-850 was similar to that of FCCP.

药物治疗后秀 丽隐杆线虫 图像分析

Protocol for Measuring Mitophagy in Hep-3B Cancer Cells 17

To begin, take 70 to 80%confluent Hep-3B cells. Remove 250 microliters of medium from each of the wells and add 50 microliters of medium with the appropriate treatment or vehicle. Add 50 microliters of medium to each well containing the staining reagent, and incubate the cells with the dye.After removing the medium and washing the cells with pre-warmed PBS, fix the cells with 200 microliters of fixing solution for 15 minutes at room temperature. Afterward, decant the fixative solution and wash briefly with 200 microliters of PBS. Finally, add 200 microliters of PBS.Keep the cells covered and protected from light at four degrees Celsius, and image within 24 hours.

测量Hep-3B癌细胞线粒体自噬的协议 17

After imaging the drug treated Hep-3B cells, open the confocal images in image J with the colocalization plugin. Open the ND file in the image J server and select the split channels option in the dialogue box. Work with green and red images.Generate duplicates of these images to keep the original image untouched by clicking on image and selecting duplicate or using the keyboard shortcut shift plus D.To reduce the background, generate another image duplicate, subtract the background with a rolling radius of 100, and select the create background option to generate an image with the given images background. Then, go to process. Click image calculator and subtract the first duplicated image from the second duplicated image.Utilize the resulting images for the colocalization analysis. To use the colocalization plugin, convert the green and red channel images to 8 bit by clicking image, selecting type, followed by 8 bit. Next, click on plugins and select colocalization.To measure the colocalization of mitochondria and lysosome signals, set the ratio to 75%the threshold red channel to 80.0, and the threshold green channel to 50.0. An 8 bit binary image containing colocalized puncta and combining the three 8 bit images in an RGB image will be generated. Convert these images to 8 bit images.To obtain the region of interest of the cells, generate an image that highlights the area of cells by selecting a colocalized point's image. To select the entire cell area, select the resultant colocalized points image and click on image, adjust threshold, and set threshold to ensure all the cell area is highlighted. To analyze the area of the cell, select analyze, click analyze particles, and measure all the particles in the image from zero to infinity, the default setting for analyze particles.To analyze the area of the colocalized mitochondria and lysosomes, select the colocalized 8 bit image. Then, select analyze. Click analyze particles and measure the summation of puncta between 0.1625 square micrometers and four square micrometers.Divide the area of the colocalized mitochondria and lysosomes by the total cell area to measure the colocalized puncta. The confocal images of Hep-3B cells displayed colocalization of mitochondria and lysosomes. VL 850 and FCCP induced significant mitophagy in the Hep-3B cells.