<meta charset="utf8"></head><body>秀丽隐杆线虫中挥发性信息素反应的提取和定量

<table><tbody><tr><td>10 cm Petri dishes</td><td>Falcon</td><td>25373-100</td><td>Falcon bacteriological Petri dish 100 x 15 mm</td></tr><tr><td>6 cm Petri dishes</td><td>Falcon</td><td>25373-085</td><td>Falcon bacteriological Petri dish 60 x 15 mm</td></tr><tr><td>&lt;em&gt;C. remanei &lt;/em&gt;(EM464)</td><td>CGC</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>centrifuge 5418</td><td>&amp;nbsp;Any brand should work.</td></tr><tr><td>Chemoattraction assay plates</td><td>Homemade solution</td><td>N/A</td><td>1.5% agar, 25 mM NaCl, 1.5 mM Tris-base, and 3.5 mM Tris-Cl</td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>57-88-5</td><td></td></tr><tr><td>Dissecting Microscope</td><td>Leica</td><td>LeicaMZ75</td><td>&amp;nbsp;Any brand should work.</td></tr><tr><td>&lt;em&gt;E. Coli&lt;/em&gt; OP50</td><td>CGC</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Koptec</td><td>64-17-5</td><td></td></tr><tr><td>&lt;em&gt;fog-2(q71)&lt;/em&gt; (JK574)</td><td>CGC</td><td></td><td></td></tr><tr><td>&lt;em&gt;him-5(e1490)&lt;/em&gt;(CB4088)</td><td>CGC</td><td></td><td></td></tr><tr><td>Household bleach</td><td>Clorox Germicidal bleach concentrated</td><td></td><td>&amp;nbsp;Bleach</td></tr><tr><td>M9 buffer</td><td>Homemade solution</td><td>N/A</td><td>3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 11.3 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O, 5 g NaCl, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L. Sterilize by autoclaving. Add 1 mL 1 M MgSO&lt;sub&gt;4&lt;/sub&gt; after cool down to room temperature.</td></tr><tr><td>Magnesium Sulfate, Anhydrous, Powder</td><td>Macron</td><td>M1063-500GM-EA</td><td></td></tr><tr><td>Microwave</td><td>TOSHIBA</td><td>N/A</td><td>&amp;nbsp;Any brand should work.</td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt;</td><td>CGC</td><td></td><td></td></tr><tr><td>NaOH</td><td>Sigma-aldrich</td><td>S318-3</td><td>1 M</td></tr><tr><td>NGM plates solution</td><td>Homemade solution</td><td>N/A</td><td>2.5 g Peptone, 18 g agar, 3 g NaCl, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L.Sterilize by autoclaving. Once the autoclave is done (2 h), wait until the temperature of the medium drops to 65 &amp;deg;C. Put on a hotplate at 65 &amp;deg;C and stir. Then add the following, waiting 5 min between each to avoid crystallization: 1 mL CaCl&lt;sub&gt;2&lt;/sub&gt; (1 M), 1 mL MgSO&lt;sub&gt;4&lt;/sub&gt; (1 M), 25 mL K&lt;sub&gt;3&lt;/sub&gt;PO4 (1 M, pH=6), 1 mL Cholesterol ( 5 mg/mL in ethanol).</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>13-374-10</td><td>Bemis Parafilm M Laboratory Wrapping Film</td></tr><tr><td>Peptone</td><td>VWR</td><td>97063-324</td><td></td></tr><tr><td>Pipet- aid</td><td>Drummond Scientific&amp;nbsp;</td><td>4-000-100</td><td>&amp;nbsp;Any brand should work.</td></tr><tr><td>Plastic paper&amp;nbsp;</td><td>Octago</td><td>Waterproof Screen Printing Inkjet Transparency Film</td><td>https://www.amazon.com/Octago-Waterproof-Transparency-Printing-Printers/dp/B08HJQWFGD</td></tr><tr><td>Potassium chloride</td><td>Sigma-aldrich</td><td>SLBP2366V</td><td></td></tr><tr><td>Potassium phosphate</td><td>Spectrum</td><td>7778-77-0</td><td></td></tr><tr><td>Pipette</td><td>Eppendorf</td><td>SKU: EPPR4331; MFG#: 2231300006</td><td>20 - 200 &amp;micro;L, 100 - 1000 &amp;micro;L, any brand should work.</td></tr><tr><td>Rotator</td><td>Labnet</td><td>SKU: LI-H5500</td><td>&amp;nbsp;Labnet H5500 Mini LabRoller with Dual Direction Rotator. Any brand should work.</td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>7647-14-5</td><td></td></tr><tr><td>sodium phosphate dibasic</td><td>Sigma-aldrich</td><td>SLCG3888</td><td></td></tr><tr><td>Tris-base</td><td>Sigma-aldrich</td><td>77-86-1</td><td></td></tr><tr><td>Tris-Cl&amp;nbsp;</td><td>Roche</td><td>1185-53-1</td><td></td></tr><tr><td>Tryptone</td><td>VWR</td><td>97063-390</td><td></td></tr><tr><td>Vortex</td><td>Scientific industries</td><td>Vortex-Genie 2</td><td>&amp;nbsp;Any brand should work.</td></tr><tr><td>WormLab system&amp;nbsp;</td><td>MBF Bioscience</td><td>N/A</td><td>https://www.mbfbioscience.com/help/WormLab/Content/home.htm; https://www.mbfbioscience.com/products/wormlab/</td></tr><tr><td>Wormpicker</td><td>Homemade&amp;nbsp;</td><td>N/A</td><td>made with platinum and glass pipet tips</td></tr></tbody></table>

volatile sex pheromone extraction and chemoattraction assay in caenorhabditis elegans

<meta charset="utf8"></head><body>作者聚焦：检查挥发性信息素对雄性 秀丽隐杆线 虫行为的影响

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

volatile-sex-pheromone-extraction-chemoattraction-assay

Research

JoVE Journal

Behavior

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

2.2K 次观看.  California Institute of Technology. 我们的研究使用趋化性测定法检查了挥发性信息素如何影响雄性秀丽隐杆线虫的行为。它旨在确定信息素产生的分子机制，其神经元机制是感知。响应信息素的个体蠕虫间变异性，再加上环境因素（如温度、湿度和光线）的影响，可能会在数据中引入大量噪声。此外，蠕虫行为的复杂性使数据解释变得复杂。应对这些挑战需要仔细的实验设计。...

视频: 作者聚焦：检查挥发性信息素对雄性 秀丽隐杆线 虫行为的影响

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

修改后的孔板 - 测量行为，认知和社会互动小鼠和大鼠

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

科研

行为学

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

基于线虫人群的化学感应优选测定的自动分析

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

使用唾液酸多糖微阵列测定在人血清中分析抗Neu5Gc IgG

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

一种简便、低成本的肌营养不良小鼠步行模型的测定方法

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

一种研究空间记忆在小鼠本能防御中作用的行为分析法

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

小龙虾心脏的连续无创测量与行为活动

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

线虫生殖道内精子引导和运动的测定

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

发育生物学

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

神经科学

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

生物学

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Source: Campbell, J. C., et al. <a href="https://www.jove.com/video/55939/a-caenorhabditis-elegans-nutritional-status-based-copper-aversion">A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay.</a> J. Vis. Exp. (2017).
This video introduces chemotaxis in the nematode C. elegans and shows a sample protocol testing chemotactic aversion to copper sulfate.

秀丽隐杆线虫趋化性测定:一种测试蠕虫化学感觉的方法

Source: Campbell, J. C., et al. A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay. J. Vis. Exp. (2017).
This video introduces ...