JLN는 MH 68347에 의해 지원되었다.

물의 미로 설정 <ol><li> 설정 물의 미로의 주요 구성 요소는 직경 6 피트 깊이 약 3 미터 정도, 라운드 풀해야합니다. 당신은 비디오 카메라로 작업을 기록하는 경우, 미로의 모든 측면이보기의 카메라의 현장 내에 있는지 확인하십시오. </li><li> 가까운 26해야 수돗물, ° C.와 물 미로를 작성 이것은 몇 시간이 걸릴 수 있으므로 사전에 잘 수행하여야한다. 그것이 26 ° C. 중 하나 정도 이내 있도록 주기적으로 물 온도를 확인 </li><li> 수영장의 중심에서 탈출 플랫폼을 놓습니다. 훈련하는 동안, 그것은, 물 위에 1 인치 노출해야합니다. 이것은 플랫폼이있다는 것을 밀고를 가르치고, 그리고 그것은 물 밖으로 얻을 수있는 방법입니다. 나중에 동물이 훈련하고 테스트하기위한 준비 후 탈출 플랫폼은 수면 아래에있을 것이며, 물이 우유 또는 무독성 페인트 불투명 만들 수 있기 때문에 표시되지 않습니다. 이제 물의 미로가 동물을 훈련 준비가 된 것입니다. </li></ol> 물의 미로에 대한 사전 교육 <ol><li> 물 미로 훈련 들어, 플랫폼은 수영장의 센터와 표면 위에 노출된 반장님에 있어야, 동물이있다는 것을 알고 있으므로. 물은 26 ° C. 중 하나도 내에 있어야합니다 </li><li> 각 동물은 세 연속 시험을 받아야합니다. 첫째, 스무 살이 초 동안 플랫폼에서 동물을 놓으십시오. </li><li> 북쪽, 남쪽, 동쪽 또는 서쪽 : 물 미로 4 시작 위치를하고 있습니다. 다음 위치 중 하나에 동물을 가져가라. 손으로 그것을 지원하고 꼬리 엔드 먼저 물에 부드럽게 내려 가져와 물에 동물을 낮춥니다. 첫번째 머리에 그런 일은하여 동물을 스트레스 받게하지 마십시오. </li><li> 동물 수영 / 60 초 최대 플랫폼을 검색하자. 처음에는 동물 나갈 방법을 찾고 수영장의 가장자리 주위에 수영 있습니다. 결국 동물은 플랫폼을 검색 배우고 올라가 것입니다. </li><li> 설치류가 플랫폼에 도착하면, 타이머를 중지하고, 시간을 기록합니다. 그것이 60 초 플랫폼을 찾을 수없는 경우, 다음 일분으로이 재판을 위해 시간을 기록합니다. 그것이 플랫폼에 도달하지 못하면 동물을 선택하지 마십시오. 그것은 플랫폼에 수영해야하는 동물을 가르치십시오. 따라서, 부드럽게 손으로 플랫폼으로 동물을 안내입니다. 동물 15 초 동안 플랫폼에 앉아 보자. 그것이 폭포거나 뛰어내리는 경우, 부드럽게 다시 안내입니다. 이것은 수영장에서 구조를 플랫폼에 체류해야하는 동물을 훈련합니다. </li><li> 각 재판에 대해 다른 방향에서 시작, 두 번 더 시도에 대해 동일한 절차를 반복합니다. </li><li> 동물이 세 시련을 완료하고 나면 수건으로 닦아 건조. 연속적으로 모든 동물에 대한 세 시범 교육 과정을 반복합니다. 그들 모두에 대한 지침에게 동일하게 유지하고, 자신의 시간을 기록합니다. </li><li> 동물 훈련 이제, 그들은 물 미로 테스트를 수행할 준비가되어 있습니다. </li></ol> 워터 미로 테스트 <ol><li> 플랫폼이 수면 아래 반장님되도록 물 미로와 실험 실험을 시작하려면, 탱크를 채우십시오. 물이 불투명하기 위해, 이외 지방 건조 우유, 또는 무독성 페인트 흰색 온도 125 밀리리터를 사용하십시오. </li><li> 조명과 물의 온도는 교육 과정에서와 동일해야합니다. 각 동물은 각각의 시작 방향 3 재판을하게됩니다, 12 시련을 받아야합니다. 각각의 시험 시간은 60 초 마지막 것입니다. 시작하기 전에 시작 방향의 순서를 선택합니다. 연속으로 두 번 같은 시작 방향을 사용하고, 또한 방향에 대해 같은 명령을 반복하지 마십시오 </li><li> 수영장 벽을 마주 동물 처리기는 물속에 동물을 배치되며, 동물 미로 작업을 수행하는 동안 다음 수영장에서 뒤로 물러나 지정된 자리에 앉아야한다고 명시하고있다. 이러한 스마트 시스템 (샌디에고 인 스트 루먼트)와 같은 동물의 동작 추적 시스템은 경로뿐만 아니라 다른 변수를 모니터링하는 데 사용할 수 있습니다. 각각의 재판을 찍고하면 저렴한, 그리고 완벽하게 적절한 대안입니다. </li><li> 이 플랫폼을 도달 때까지 동물을 모니터하고 그것이 찍은 시간을 기록합니다. 동물이 60 초 플랫폼을 도달하지 않는 경우, 핸들러는 교육에서와 같이, 플랫폼으로 안내합니다. 어느 쪽이든, 동물 10 초 동안 앉아, 다음을 건조하고 개최 케이지로 동물을 반환하자. </li><li> 동물 처리기는 각 재판 중에 같은 지정된 장소에 반환과 함께 모든 12 시련 각 동물을 계속합니다. 테스트의 순서는 다음과 같아야합니다 모든 동물들에게 시험 1, 모든 동물에 대한 재판이 모든 동물에 대한 재판 3, 등 최소한 2 분 간 재판 간격이 있어야합니다. 정기적으로, 수영장을 청소 플랫폼이 자리에 있는지 확인하고, 물의 온도가 동일한지 확인합니다. </li><li> 모든 동물 12 시련을 마친 후, 각 뭐 한 프로브 재판을 수행합니다ich 플랫폼은 수영장에서 제거됩니다. 프로브 재판은 플랫폼 위치의 동물의 이해를 확인하기 위해 수행하고, 동물이 그것은 플랫폼이 없습니다 발견되면 다음과 그 전략을 관찰합니다. 핸들러는 북쪽에서 시작하는 동물을 발표할 예정이다. 동물이 30 초 동안 수영장의 중심을 교차 횟수를 기록합니다. </li><li> 모든 프로브 재판이 완료되면, 동물에서 건조하고 수영장 드레인. </li></ol>

<ol>
	<li>de Bruin, J. P., Swinkels, W. A., de Brabander, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+learning+of+rats+in+a+Morris+water+maze:+involvement+of+the+medial+prefrontal+cortex.">Response learning of rats in a Morris water maze: involvement of the medial prefrontal cortex.</a> Behav. Brain Res. 85, 47-55 (1997).</li><li>Redish, A. D., Touretzky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+hippocampus+in+solving+the+Morris+water+maze.">The role of the hippocampus in solving the Morris water maze.</a> Neural. Comput. 1, 73-111 (1998).</li><li>D'Hooge, R., De Deyn, P. 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The authors have nothing to disclose.

물 미로의 작업은 모리스 5 개발했습니다. 이 작업은 메모리 참조 메모리와 작업 전략 6 일을 조사하기 위해 다양한 방법으로 변경할 수 있습니다. pretraining, 그리고 하루에 시험 : 여기에 설명된 절차는 물 미로의 다른 버전에서 편차를 나타내는 중요한 두 변수가 포함되어 있습니다. Pretraining - 해마는 공간 7-8 / 관계형 메모리에 참여합니다. 물 미로는 특히 공간 메모리...

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<td>SMART System</td>
<td>San Diego Instruments</td>
<td>&nbsp;</td>
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<td>This system is not a requirement &#x2013; there are many other behavioral systems on the market.</td>
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morris water maze experiment

모리스 물 미로 널리 공간 기억과 학습을 연구하는 데 사용됩니다. 동물은 가루가 아닌 지방 우유 또는 그들이 숨겨진 탈출 플랫폼으로 수영을해야합니다 무독성 페인트 온도와 불투명 컬러는 물을 수영장에서 배치됩니다. 그들이 불투명 물에 있기 때문에, 동물 플랫폼을 볼 수 없으며, 탈출 경로를 찾을 수 향기에 의존 수 없습니다. 대신, 그들은 외부 / 엑스트라 미로의 단서에 의존해야합니다. 동물들이 작업을 더욱 친숙 해짐에 따라, 그들은 더 빨리 플랫폼을 찾을 수 있습니다. 1984 년 리처드 G. 모리스에 의해 개발,이 패러다임은 행동 신경 과학의 &quot;금 표준&quot;중 하나가되었습니다.

Watch this Scientific Journal Video about Morris Water Maze Experiment at JoVE.com

Morris Water Maze Experiment

모리스 물 미로는 행동 작업에 해마의 참여를 문서화하는 데 사용하는 잘 접수 도구입니다.

모리스 워터 메이즈 실험

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Research

JoVE Journal

Biology

56.1K 조회수.  Michigan State University (MSU). 모리스 물 미로는 행동 작업에 해마의 참여를 문서화하는 데 사용하는 잘 접수 도구입니다.

비디오: Morris Water Maze Experiment

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

인공 다이어트 실험을 사용하여 Aphids에 니코틴 공차 테스트

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

연구

생물학

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

마우스에 대한 고가 플러스 메이즈

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

이미징 CA의 기술 2 + 인간 정자의 시그널링

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

C2C12 Myoblast 세포를 사용하여 Bioenergetic 프로필 실험

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

토양에 미생물 잔류 결정을위한 Aldononitrile 아세트산 Derivati​​zed 글루코사민과 Muramic 산성의 GC 기반 탐지

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

도입 단백 분해 효소와 산 촉매 라벨링 워크 플로<sup&gt; 18</sup&gt; O 강화 물

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

주름 식민지 개발을 사용하여 Biofilm 형성을 평가하기 위해 세미 양적 접근

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

분리 및 문화 신생아 마우스 심근

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

마우스 표피 각질 세포와 체외 클로노겐 문화의 격리

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...