<meta charset="utf8"></head><body>去除斑马鱼鳞片用于培养和骨骼生物学研究

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<table><tbody><tr><td>0.2 mL tubes</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>37% HCl&amp;nbsp;</td><td>EMD</td><td>HX0603-4</td><td>For pH stabilization and prepration of PRS</td></tr><tr><td>Concave slide</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>DAPI</td><td>Vectashield</td><td>H-1200</td><td>For cell nuclei visualization</td></tr><tr><td>Diazonium salt (Fast Blue B)</td><td>Sigma</td><td>D9805</td><td>For AP substrate solution</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) powder&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>D5523</td><td>For scale culture media&amp;nbsp;</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (MS222)&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>E10521</td><td>For preparation of 0.1% MS222</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>F4135</td><td>For scale culture media&amp;nbsp;</td></tr><tr><td>Fluorescence microscope</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Glacial acetic acid&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>A38 212</td><td>For acetate buffer</td></tr><tr><td>Glycerol</td><td>VWR</td><td>BDH1172-4LP</td><td>For 80% glycerol - storage scales</td></tr><tr><td>HEPES&amp;nbsp;</td><td>ThermoFisher&amp;nbsp;</td><td>15630106</td><td>For scale culture media&amp;nbsp;</td></tr><tr><td>Hot plate</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>KCl&amp;nbsp;</td><td>Sigma</td><td>P217-10</td><td>For preparation of PBS</td></tr><tr><td>Lysotracker</td><td>ThermoFisher</td><td>L7528</td><td>For lysosomes&amp;nbsp; visualization</td></tr><tr><td>Maleic acid</td><td>Sigma</td><td>M0375</td><td>For Tris-Maleate buffer&amp;nbsp;</td></tr><tr><td>Micropipette</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>N,N-dimethylformamide</td><td>Sigma</td><td>319937</td><td>For AP substrate solution</td></tr><tr><td>N,N-dimethylformamide&amp;nbsp;</td><td>Sigma</td><td>319937</td><td>For Enzyme Substrate Solution</td></tr><tr><td>Na2HPO4&amp;nbsp;</td><td>EMD</td><td>SX0720-1</td><td>For preparation of PBS</td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>S9888</td><td>For preparation of PBS</td></tr><tr><td>NaNO&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>S-2252</td><td>For 4% NaNO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>NaOH&amp;nbsp;</td><td>Sigma</td><td>S5881</td><td>For preparation of 4% PFA</td></tr><tr><td>NaOH&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>S5881</td><td>For scale fixation and pH stabilization</td></tr><tr><td>Napthol-AS-TR-phosphate</td><td>Sigma</td><td>N6125</td><td>For AP substrate solution</td></tr><tr><td>Napthol-AS-TR-phosphate&amp;nbsp;</td><td>Sigma</td><td>N6125</td><td>For Enzyme Substrate Solution</td></tr><tr><td>Pararosaniline hydrochloride&amp;nbsp;</td><td>Sigma</td><td>P3750</td><td>For PRS</td></tr><tr><td>Penicillin-streptomycin 5000 units penicillin and 5mg streptomyocin/ml</td><td>ThermoFisher&amp;nbsp;</td><td>15140122</td><td>For scale culture media&amp;nbsp;</td></tr><tr><td>Personal protective equipment (PPE): disposable nitrile gloves, safety glasses/splash goggles and lab coat.</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>PFA&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P6148</td><td>For scale fixation</td></tr><tr><td>Sodium acetate&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>S2889</td><td>For acetate buffer</td></tr><tr><td>Standard compound microscope</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Stir plate</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Tartaric acid</td><td>Sigma</td><td>T6521</td><td>For Tartrate buffer</td></tr><tr><td>Tris base</td><td>Roche</td><td>03 118 142 001</td><td>For Tris-Maleate buffer&amp;nbsp;</td></tr></tbody></table>

in vitro culturing technique for studying cellular dynamics in zebrafish scales

<meta charset="utf8"></head><body>作者聚焦：利用斑马鱼鳞片推进骨细胞活性研究

用于研究斑马鱼鳞片细胞动力学的体外培养技术

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without ...

in-vitro-culturing-technique-for-studying-cellular-dynamics-zebrafish

Research

JoVE Journal

Developmental Biology

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

2.8K 次观看.  Mount Saint Vincent University. 我们的研究探讨了骨组织环境干扰的影响。我们对了解模拟微重力和振动对成人和骨骼发育的影响特别感兴趣。我们使用斑马鱼作为首选的模式生物，因为它为生物研究提供了许多优势。与以前的方案相比，我们的方案具有优势，因为它限制了对二氧化碳培养箱的需求，并且它使用标准实验室设备。我们的实验室正试图解开不同类型骨细胞在受...

视频: 作者聚焦：利用斑马鱼鳞片推进骨细胞活性研究 

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

胚胎和肝脏斑马鱼细胞系球状体的3D培养方法

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

环境科学

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

生物学

在体内再生过程中斑马鱼软骨鳞片的收获和可视化

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new therapeutics that could ease the burden on healthcare systems. As these diseases are complex, it is difficult and expensive to accurately model bone pathophysiology in a lab setting. The challenge for the field is to establish a cost-effective, biologically relevant platform for modeling bone disease that can be used to test potential therapeutic compounds. Such a platform should ideally allow dynamic visualization of cell behaviors of bone-building osteoblasts and bone-degrading osteoclasts acting in their mineralized matrix environment. Zebrafish are increasingly used as models due to the availability of genetic tools, including transgenic reporter lines, and the fact that some skeletal tissues (including the scales) remain translucent to adulthood, allowing dynamic imaging options. Since zebrafish scales have both osteoblasts and osteoclasts and are highly abundant, they provide an easily accessible and abundantly available resource of independent bone units. Moreover, once removed, adult zebrafish scales fully regenerate, therefore offering a way to study the spatiotemporal growth of mineralized tissue in vivo. Here, we detail protocols for harvesting and tracking the regeneration of the scales. Lastly, a protocol for stable culture of scales ex vivo for a week and following the healing response after controlled damage to the mineralized matrix of the scale over time is also presented.

作者聚焦:使用鱼鳞片进行骨重塑研究 – 离体成像和解剖细胞相互作用的进展

This protocol describes the harvesting and visualization of elasmoid scales of zebrafish during in vivo regeneration. In addition, the ex vivo culture of these scales for up to 7 days after harvest is presented.

斑马鱼鳞片再生 在多多 和 离体 鳞片培养中

Skeletal diseases are often complex in their etiology and affect millions of people worldwide. Due to the aging population, there is a need for new ...

用于研究斑马鱼鳞片细胞动力学的体外培养技术

副本

摘要

探索更多视频

此视频中的章节

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

技术目标显微注射到显影

分离和斑马鱼胚胎单细胞的表征

观察有丝分裂和动力学在现场斑马鱼胚胎

生物传感运动神经元膜电位在活斑马鱼胚胎

胚胎和肝脏斑马鱼细胞系球状体的3D培养方法

斑马鱼原代细胞的培养与转染

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

斑马鱼鳞片再生在多多和离体鳞片培养中

用于研究斑马鱼鳞片细胞动力学的体外培养技术

副本

摘要

探索更多视频

此视频中的章节

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

技术目标显微注射到显影

分离和斑马鱼胚胎单细胞的表征

观察有丝分裂和动力学在现场斑马鱼胚胎

生物传感运动神经元膜电位在活斑马鱼胚胎

胚胎和肝脏斑马鱼细胞系球状体的3D培养方法

斑马鱼原代细胞的培养与转染

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

斑马鱼鳞片再生 在多多 和 离体 鳞片培养中

斑马鱼鳞片再生在多多和离体鳞片培养中