propagation, differentiation, and preparation of sh-sy5y cells for live cell imaging

使用 STED 显微镜揭示神经元中的线粒体超微结构

Mitochondria are eukaryotic organelles of endosymbiotic origin that are responsible for regulating several key cellular processes, including intermediary metabolism and ATP production, ion homeostasis, lipid biosynthesis, and programmed cell death (apoptosis). These organelles are topologically complex, containing a double membrane system that establishes multiple subcompartments1 (Figure 1A). The outer mitochondrial membrane (OMM) interfaces with the cytosol and establishes direct inter-organelle contacts2,3. The inner mitochondrial membrane (IMM) is an energy-conserving membrane that maintains ion gradients stored primarily as an electric membrane potential (&#916;&#936;m) to drive ATP synthesis and other energy-requiring processes4,5. The IMM is further subdivided into the inner boundary membrane (IBM), which is closely appressed to the OMM, and protruding structures called cristae that are bound by the cristae membrane (CM). This membrane delineates the innermost matrix compartment from the intracristal space (ICS) and the intermembrane space (IMS).
Mitochondria have a dynamic morphology based on continuous and balanced processes of fission and fusion that are governed by mechanoenzymes of the dynamin superfamily6. Fusion allows for increased connectivity and formation of reticular networks, whereas fission leads to mitochondrial fragmentation and enables the removal of damaged mitochondria by mitophagy7. Mitochondrial morphology varies by tissue type8 and developmental stage9 and is regulated to allow cells to adapt to factors including energetic needs10,11 and stressors12. Standard morphometric features of mitochondria, such as the extent of network formation (interconnected vs. fragmented), perimeter, area, volume, length (aspect ratio), roundness, and degree of branching, can be measured and quantified by standard optical microscopy because the sizes of these features are greater than the diffraction limit of light (~200 nm)13.
Cristae architecture defines the internal structure of mitochondria (Figure 1B). The diversity of cristae morphologies can be broadly categorized as flat (lamellar or discoidal) or tubular-vesicular14. All cristae attach at the IBM through tubular or slot-like structures termed cristae junctions (CJs) that can serve to compartmentalize the IMS from the ICS and the IBM from the CM15. Cristae morphology is regulated by key protein complexes of the IMM, including (1) the mitochondrial contact site and cristae organizing system (MICOS) that resides at CJs and stabilizes IMM-OMM contacts16, (2) the optic atrophy 1 (OPA1) GTPase that regulates cristae remodeling17,18,19, and (3) F1FO ATP synthase that forms stabilizing oligomeric assemblies at cristae tips (CTs)20,21. In addition, the IMM is enriched in nonbilayer phospholipids phosphatidylethanolamine and cardiolipin that stabilize the highly curved IMM22. Cristae are also dynamic, demonstrating morphological changes under various conditions, such as different metabolic states23,24, with different respiratory substrates25, under starvation and oxidative stress26,27, with apoptosis28,29, and with aging30. Recently it has been shown that cristae could undergo major remodeling events on a timescale of seconds, underscoring their dynamic nature31. Several features of cristae can be quantified, including dimensions of structures within individual cristae (e.g., CJ width, crista length, and width) and parameters that relate individual crista to other structures (e.g., intra-cristae spacing and cristae incident angle relative to the OMM)32. These quantifiable cristae parameters show a direct correlation with function. For instance, the extent of mitochondrial ATP production is positively related to the abundance of cristae, quantified as cristae density or cristae number normalized to another feature (e.g., cristae per OMM area)33,34,35. Because IMM morphology is defined by nanoscale features, it comprises mitochondrial ultrastructure, which requires imaging techniques that provide resolution greater than the light diffraction limit. As described below, such techniques include electron microscopy and super-resolution microscopy (nanoscopy).
The neural and glial cells of the central nervous system (CNS) are particularly reliant on mitochondrial function. On average, the brain constitutes only 2% of the total body weight, but utilizes 25% of the total body glucose and accounts for 20% of body oxygen consumption, making it vulnerable to impairments in energy metabolism36. Progressive neurodegenerative diseases (NDs), including Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington&#39;s disease (HD), multiple sclerosis (MS), and Parkinson&#39;s disease (PD), are some of the most extensively studied pathologies to date, with research efforts ranging from understanding the molecular underpinnings of these diseases to seeking potential therapeutic prevention and interventions. NDs are associated with increased oxidative stress originating in part from reactive oxygen species (ROS) generated by the mitochondrial electron transport chain (ETC)37, as well as altered mitochondrial calcium handling38 and mitochondrial lipid metabolism39. These physiological alterations are accompanied by noted defects in mitochondrial morphology that are associated with AD40,41,42,43,44, ALS45,46, HD47,48,49, MS50, and PD51,52,53. These structural and functional defects can be coupled by complex cause-effect relationships. For example, given that cristae morphology stabilizes OXPHOS enzymes54, mitochondrial ROS are not only generated by the ETC, but they also act to damage the infrastructure in which the ETC resides, promoting a feed-forward ROS cycle that enhances susceptibility to oxidative damage. Furthermore, cristae disorganization has been shown to trigger processes such as mitochondrial DNA (mtDNA) release and inflammatory pathways connected to autoimmune, metabolic, and age-related disorders55. Therefore,&#160;analysis of&#160;mitochondrial structure is key to a full understanding of NDs and their molecular underpinnings.
Popular methods of viewing cristae, including transmission electron microscopy, electron tomography and cryo-electron tomography (cryo-ET), and X-ray tomography, in particular cryo-soft X-ray tomography, have revealed important findings and work with a variety of sample types56,57,58,59,60. Despite recent advancements toward better observation of organellar ultrastructure, these methods still come with the caveat of requiring sample fixation and, therefore, cannot capture real-time dynamics of cristae directly. Super-resolution fluorescence microscopy, particularly in the forms of structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), expansion microscopy (ExM), and stimulated emission depletion (STED) microscopy, have become popular ways of viewing structures requiring resolution below the diffraction limit that constrains classical methods of optical microscopy. When ExM is used in conjunction with another super-resolution technique, the results are impressive, but the sample must be fixed and stained in a gel61. By comparison, SIM, PALM/STORM, and STED have all been successfully used with live samples, and new and promising dyes that generally stain the IMM provide a novel and easy approach for live imaging of mitochondria cristae dynamics62,63,64,65,66. Recent advancements in live dyes for STED imaging have improved dye brightness and photostability, and these dyes target the IMM at a higher degree of specificity than their predecessors. These developments allow the collection of long-term timelapse and z-stack experiments with super-resolution imaging, opening the door to better live cell analysis of mitochondrial ultrastructure and dynamics.
Herein, protocols for live cell imaging of undifferentiated and differentiated SH-SY5Y cells stained with the PKmito Orange (PKMO) dye using STED63 are provided. The SH-SY5Y cell line is a thrice subcloned derivative from the parental cell line, SK-N-SH, generated from a bone marrow biopsy of metastatic neuroblastoma67,68,69,70. This cell line is a commonly used in vitro model in ND research, particularly with diseases such as AD, HD, and PD, in which mitochondrial dysfunction is heavily implicated10,43,71,72,73. The ability to differentiate SH-SY5Y cells into cells with a neuron-like phenotype through manipulating culture media has proven a suitable model for neuroscience research without relying on primary neuronal cells10,74. In this protocol, retinoic acid (RA) was added to the cell culture medium to induce the differentiation of SH-SY5Y cells. RA is a vitamin A derivative and has been shown to regulate the cell cycle and promote the expression of transcription factors that regulate neuronal differentiation75. A protocol for culturing and live cell imaging of neurons isolated from the rat hippocampus is also provided. The hippocampus has been shown to be affected by mitochondrial degeneration and, along with the cortex, plays an important role in aging and ND76,77,78,79,80.

作者聚焦：解码线粒体衰老 

使用活细胞STED成像可视化神经元细胞模型中的线粒体内膜超微结构

Our research program focuses on structural and functional aspects of mitochondria, particularly as they relate to aging related dysfunction. To this end, we use a broad array of biophysical, biochemical, and structural techniques to explore the basic mechanisms of mitochondrial aging and to develop therapeutic interventions to preserve and restore mitochondrial health. Mitochondrial function and structure are inextricably related.Standard light microscopy techniques are suitable for measuring large scale mitochondrial features like basic morphology and network structures. Analyzing mitochondrial ultra structure or features that require higher resolution than afforded by traditional optical microscopy is typically done using electron microscopy or tomography on fixed samples. Super resolution microscopy is a fluorescence based imaging technique that measures features beyond the diffraction limit.For mitochondria, this includes measurements of nano scale protein distribution and cristae architecture. The stead imaging protocol described here allows researchers to measure the detailed structure of the inner membrane in living cells. Live cell imaging allows us to observe and quantitatively analyze complex features of cristae under physiologically relevant conditions without the need for sample fixation.This will give researchers novel insights into the dynamic changes that occur in ultra structural features and their temporal responses to stressors and pharmacological compounds.

Watch this Scientific Journal Video about Propagation, Differentiation, and Preparation of SH-SY5Y Cells for Live Cell Imaging at JoVE.com

Propagation, Differentiation, and Preparation of SH-SY5Y Cells for Live Cell Imaging  

用于活细胞成像的SH-SY5Y细胞的增殖、分化和制备

首先，在FBS中快速解冻SH-SY5Y细胞的冷冻储备液。补充 10% DMSO，并用预热培养基稀释 10 倍。然后离心细胞并除去上清液。将细胞沉淀重悬于5毫升预热培养基中，并在T25烧瓶中接种细胞。为了制备涂有PDL的盖玻片，将每毫升50微克的600微升PDL溶液涂在无菌腔盖玻片的每个孔中。添加到每个孔中的PDL体积因孔大小而异。将盖玻片在室温下孵育一小时。孵育后，除去PDL溶液，用1.8毫升蒸馏水冲洗盖玻片三次。让涂层室风干两小时。一旦SH-SY5Y细胞达到80%至90%的汇合度，通过添加胰蛋白酶溶液将其解离。通过添加预热的DMEM培养基来中和胰蛋白酶。离心细胞悬液并将沉淀重新悬浮在DMEM中。在自动细胞计数仪上对细胞进行计数。接下来，将细胞以 1.5 乘以 10 到每平方厘米第四个细胞的密度接种到 PDL 涂层腔盖玻璃上。在第一天和第三天，用分别补充有 5% 或 2% FBS、1% 抗生素抗真菌剂和相同添加剂体积的 10 μmol RA 或 95% 乙醇的 DMEM 替换培养基，作为分化的载体对照。在预热的无苯红DMEM中制备PKMO储备液，并补充有2%或10%FBS（取决于分化状态）、1%抗生素抗真菌药和20毫摩尔肝炎。第六天，用成像介质冲洗细胞两次，并将细胞与PKMO溶液在37摄氏度和5%二氧化碳下孵育30分钟。染色后，用预热的成像介质冲洗细胞三次。对于最后的洗涤，将细胞在 37 摄氏度和 5% 二氧化碳下孵育 30 分钟。孵育后，用含有 20 毫摩尔 hepes 的新鲜预热成像培养基替换最终洗涤液。

propagation-differentiation-preparation-sh-sy5y-cells-for-live-cell

Research

JoVE Journal

Neuroscience

Propagation, Differentiation, and Preparation of SH-SY5Y Cells for Live Cell Imaging

528 次观看.  University of Connecticut. 首先，在FBS中快速解冻SH-SY5Y细胞的冷冻储备液。补充 10% DMSO，并用预热培养基稀释 10 倍。然后离心细胞并除去上清液。将细胞沉淀重悬于5毫升预热培养基中，并在T25烧瓶中接种细胞。为了制备涂有PDL的盖玻片，将每毫升50微克的600微升PDL溶液涂在无菌腔盖玻片的每个孔中。添加到每个孔中的PDL体积因孔大小而异。将盖玻片在室温下孵育一小时。孵育后，除去PDL溶?...

视频: Propagation, Differentiation, and Preparation of SH-SY5Y Cells for Live Cell Imaging  

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

科研

神经科学

To begin, rapidly thaw a frozen stock of SH-SY5Y cells in FBS. Supplemented with 10%DMSO and dilute tenfold with pre-warmed media. Then centrifuge the cells and remove the supernatant.Re-suspend the cell pellet in five milliliters of pre-warmed media and seed cells in a T25 flask. To prepare cover slips coated with PDL apply 600 microliters of 50 micrograms per milliliter PDL solution to each well of sterile chambered cover slips. The volume of PDL added to each well varies based on well size.Incubate the cover slips set room temperature for one hour. After incubation, remove the PDL solution and rinse the cover slip thrice with 1.8 milliliters of distilled water. Allow the coated chamber to air dry for two hours.Once the SH-SY5Y cells reach 80 to 90%confluency, dissociate them by adding trypsin solution. Neutralize the trypsin by adding a pre-warmed DMEM medium. Centrifuge the cell suspension and re-suspend the pellet in DMEM.Count the cells on an automated cell counter. Next, seed the cells at a density of 1.5 times 10 to the fourth cells per square centimeter onto the PDL coated chambered cover glass. On day one and day three, replace the medium with DMEM supplemented with either five or 2%FBS respectively, 1%antibiotic antimycotic, and either 10 micromolar RA or 95%ethanol of the same additive volume to serve as the vehicle control for differentiation.Prepare a PKMO stock in pre-warmed Phenyl red free DMEM supplemented with two or 10%FBS dependent on differentiation state, 1%antibiotic antimycotic, and 20 millimolar hepes. On day six, rinse the cells twice with imaging media and incubate cells with the PKMO solution at 37 degrees celsius and 5%carbon dioxide for 30 minutes. After staining, rinse the cells thrice with pre-warmed imaging media.For the final wash, incubate the cells for 30 minutes at 37 degrees celsius, and 5%carbon dioxide. After incubation, replace the final wash with fresh pre-warmed imaging media containing 20 millimolar hepes.

Imaging SH-SY5Y Cells by STED Microscopy and Subsequent Analysis of Mitochondrial Ultrastructure

Open the LightBox software for image acquisition. To select the laser and filter sets, use parameters for an orange dye by selecting the dye used in the staining from the dye list. Using an overview, click on live to focus the cells.Once the cells are in focus, adjust the confocal acquisition settings. Next, select the rectangular ROI button and create a region of interest around a mitochondria of interest by clicking and dragging to shape the region. To adjust detector gating for stimulated emission depletion or stead acquisition, next to the general menu, select the gating menu or click and hold to add the menu to the view.For stead acquisition, set the excitation laser to 15 to 20%and the stead depletion laser to 20 to 25%with 10 line accumulations. Use a pixel dwell time of four microseconds and a pixel size of 20 to 25 nanometers. Perform time lapse by selecting the time dropdown menu, then setting the number of iterations to five and a time interval of 25 or 30 seconds.A Z-stack can be taken using the volume option and adjusting the desired Z volume range and step size. Open stead image deconvolution software to deconvolute raw stead images with the software algorithm. After ensuring microscopic parameters are correct, select the express button and set the deconvolution type too fast, standard, aggressive, or conservative for varying degrees of deconvolution power.Execute the deconvolution. Save the images in ICS2 format. In image J, click on file then open to open the ICS2 files from the deconvolution software.Select plugins, then segmentation followed by trainable Weka segmentation to open the deconvoluted stead images in the trainable Weka segmentation plugin. In the segmentation settings, select the Gaussian blur, membrane projections and sobel filter features. Label one class as cristae and the other as background.Next, draw a line over the structure to assign to either class. Then select the add button on the right hand side for either cristae or background. Then select the train classifier button on the left hand side to generate a map based on the information provided to the plugin.Click the save classifier button to save the classifier settings for future segmentation. Use the cristae probability map to threshold the image in image J to generate a binary mask, and then go to analyze, then analyze particles. Finally, draw a multi-point line, adjust the line thickness to several pixels wide and spline the line to fit the mitochondria.In this study, imaging of mitochondria in undifferentiated, retinoic acid differentiated SH-SY5Y cells with time-lapse imaging as shown. The deconvoluted stead images can be used to determine cristae periodicity in a given area and cristae size and shape measurements.