JLNは、MH 68347によってサポートされていました。

水迷路の設定<ol><li>セットアップ水迷路の主なコンポーネントは、直径6フィート、深さ約3フィートほど、円形プールでなければなりません。あなたがビデオカメラを使用してタスクを記録している場合は、迷路のすべての辺がカメ​​ラの視野内であることを確認してください。 </li><li>近くに26〜でなければならない水道水、℃の水迷路を埋めるこれには数時間かかる場合がありますので、事前によく行われる必要があります。それは26℃の1度以内になるように、定期的に水の温度を確認してください</li><li>プールの中央にエスケーププラットフォームを置きます。訓練中に、それは1インチ水の上、公開される必要があります。これは、プラットフォームがあることをラットを教え、そしてそれは水から抜け出すための方法であること。 Later、動物が訓練を受け、テストの準備ができた後、エスケーププラットフォームは、単に水の表面の下になり、水はミルクまたは非毒性塗料と不透明されているので、表示されません。 今、水迷路は、動物を訓練するための準備ができています。 </li></ol>水迷路での事前研修<ol><li>水迷路のトレーニングについては、プラットフォームがプールの中心と表面上に露出さ1インチになるはず、動物はそれがあることを知っているので。水は26℃の1度以内でなければなりません</li><li>各動物には、3つの連続した​​試験を受けることになる。最初に、20秒のためにプラットフォーム上で動物を置きます。 </li><li>北、南、東、または西：水迷路は、4日からポジションを持っています。これらの位置のいずれかに動物を取る。あなたの手でそれをサポートする、最初の水のテールエンドに優しく、それをダウンさせることで水の中に動物を下ろします。第一の頭の中でそれをダンクで動物を強調しないでください。 </li><li>動物の泳ぎ/ 60秒の最大のプラットフォームを検索してみましょう。最初は、動物が出な方法を探して、プールの端の周りに泳ぐことができる。最終的に、動物は、プラットフォームの検索と登ることを学習します。 </li><li>げっ歯類は、プラットフォームに達すると、タイマーを停止し、時間を記録。それは60秒でプラットフォームを見つけられない場合、その後1分として、この試験のための時間を記録する。それはプラットフォームに到達するために失敗した場合、動物を持ち上げないでください。それはプラットフォームに泳ぐ必要があることを動物を教える。したがって、そっと手でプラットフォームに動物を導く。動物は、15秒間のプラットフォームの上に座ってみましょう。落ちたり、倒れたり、オフにジャンプする場合、静かにそれを導く。これは、プールから救出されるプラットフォーム上にとどまる必要があることを動物を訓練します。 </li><li>各試験ごとに異なる​​方向から始まる、2つの試験のための同じ手順を繰り返します。 </li><li>動物はすべて3つの試験を完了した後、タオルで拭き取って乾かす。連続してすべての動物の3つのトライアルのトレーニングプロセスを繰り返します。それらのすべての方向を同じに保つ、そしてその時間を記録する。 </li><li>今の動物が訓練を受けていること、彼らは、水迷路テストを実行する準備が整いました。 </li></ol>水迷路試験<ol><li>プラットフォームは、水の表面の下に1インチになるように水迷路を使った実験的臨床試験を開始するには、ガソリンを満タンにする。水が不透明にするため、脱脂粉乳、または非毒性の白テンペラ絵の具の125ミリリットルを使用してください。 </li><li>照明と水の温度は、トレーニングのプロセスと同じでなければなりません。各動物は、それぞれの開始の方向のための3試験先となる、12試験を受けることになる。各試験は60秒続きます。開始する前に、出発方向の順序を選択します。行中で、同じスタートの方向を使用し、また方向のいずれかの同じ順序を繰り返すことはしないでください</li><li>プールの壁に直面して、動物のハンドラは、水に動物を配置し、その後、プールからしりぞくと動物が迷路のタスクを実行している間、指定された場所に座ります。このようなSMARTシステム（サンディエゴの楽器）などの動物の行動追跡システムは、パスだけでなく、他の変数を監視するために使用することができます。各試験をビデオテープに録画するより安価な、そして別の、完全に適切です。 </li><li>それはプラットフォームに到達するまで、動物を監視し、それにかかった時間を記録。動物は60秒でプラットフォームに到達しない場合、ハンドラは訓練のように、プラットフォームにご案内いたします。いずれにせよ、動物は10秒間座って、そして次にそれを乾かし、保持ケージに動物を返すようにしてください。 </li><li>動物のハンドラは、各試行の間に同一の指定場所に戻ると、全12試験の各動物に進みます。テストの順番は次のようになります。すべての動物のための試験1、すべての動物のためのトライアル2、すべての動物のための裁判3など、少なくとも2分間の間トライアル間隔があるはずです。定期的に、プールをきれいに、プラットフォームの場所にある、と水の温度が同じであることをチェックしてください。 </li><li>すべての動物は12試験を完了した後、彼らはそれぞれのWHで、一つのプローブ試験を実行しますICHプラットフォームはプールから削除されます。プローブのトライアルは、プラットフォームの位置の動物の理解を検証するため、そしてそれは、プラットフォームがない検出したときに動物が続くことの戦略を観察しています。ハンドラは、北から始まる動物を解放します。動物は、30秒の間にプールの中央を横切る回数を記録します。 </li><li>すべてのプローブの臨床試験が完了したら、動物をふいて乾かすと、プールを排出。 </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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the Morris water maze in the study of learning and memory.</a> Brain Res. Rev. 36, 60-90 (2001).</li><li>Kallai, J., Makany, T., Karadi, K., Jacobs, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+orientation+strategies+in+Morris-type+virtual+water+task+for+humans.">Spatial orientation strategies in Morris-type virtual water task for humans.</a> Behav. Brain Res. 159, 187-196 (2005).</li><li>Morris, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+of+a+water-maze+procedure+for+studying+spatial+learning+in+the+rat.">Developments of a water-maze procedure for studying spatial learning in the rat.</a> J. Neurosci. Methods. 11, 47-60 (1984).</li><li>Brandeis, R., Brandys, Y., Yehuda, 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+use+of+the+Morris+Water+Maze+in+the+study+of+learning+and+memory.">The use of the Morris Water Maze in the study of learning and memory.</a> Int. J. Neurosci. 48, 26-69 (1989).</li><li>Kesner, R. P., Exans, R. B., Hunt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+evidence+in+support+of+the+neurobiological+bases+of+an+attribute+model+of+memory:+role+of+the+hippocampus.">Further evidence in support of the neurobiological bases of an attribute model of memory: role of the hippocampus.</a> Int. J. Neurosci. 21-22, 21-22 (1987-8).</li><li>Jarrard, L. E., Okaichi, H., Steward, O., Goldschmidt, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+hippocampal+connections+in+the+performance+of+place+and+cue+tasks:+comparisons+with+damage+to+the+hippocampus.">On the role of hippocampal connections in the performance of place and cue tasks: comparisons with damage to the hippocampus.</a> Behav. Neurosci. 98, 946-954 (1984).</li><li>Hodges, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+procedures:+the+radial-arm+and+water+maze+compared.">Maze procedures: the radial-arm and water maze compared.</a> Cogn. Brain Res. 3, 167-181 (1996).</li><li>Shumake, J., Gonzalez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+systems+underlying+susceptibility+to+helplessness+and+depression.">Brain systems underlying susceptibility to helplessness and depression.</a> Behav. Cogn. Neurosci. Rev. 2, 198-221 (2003).</li><li>Owen, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+the+menstrual+cycle.">Physiology of the menstrual cycle.</a> Am. J. Clin. Nutr. 28, 333-338 (1975).</li><li>Everett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+reproduction+in+the+female+rat.">Neurobiology of reproduction in the female rat.</a> Monogr. Endocrinol. 32, 1-133 (1989).</li><li>Warren, S. G., Humphreys, A. G., Juraska, J. M., Greenough, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTP+varies+across+the+estrous+cycle:+enhanced+synaptic+plasticity+in+proestrus+rats.">LTP varies across the estrous cycle: enhanced synaptic plasticity in proestrus rats.</a> Brain Res. 703, 26-30 (1995).</li><li>Warren, S. G., Juraska, 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=Spatial+and+nonspatial+learning+across+the+rat+estrous+cycle.">Spatial and nonspatial learning across the rat estrous cycle.</a> Behav. Neurosci. 111, 259-266 (1997).</li></ol>

The authors have nothing to disclose.

水迷路タスクは、モリス5で開発した。このタスクは、メモリ参照のメモリとタスクの戦略6を作業調査するためにさまざまな方法で変更することができます。 pretraining、および1日のうちにテスト：ここで説明する手順では、水迷路の他のバージョンからの偏差を表す2つの重要な変数が含まれています。 Pretraining -海馬は7から8の空間/リレーショナル記憶に関与して...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SMART System</td>
<td>San Diego Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>This system is not a requirement &#x2013; there are many other behavioral systems on the market.</td>
</tr>		</tbody>
		</table>
		</div>

morris water maze experiment

モリス水迷路は、広く空間記憶や学習を研究するために使用されます。動物は、脱脂粉乳またはそれらが隠されたエスケーププラットフォームに泳ぐ必要が無毒なテンペラ絵の具、と不透明に着色された水のプールに配置されます。彼らは不透明な水の中にあるので、動物は、プラットフォームを見ることができない、と逃げ道を見つけるために香りに頼ることはできません。その代わり、彼らは外部/エクストラ-迷路の手がかりに依存する必要があります。動物がタスクに慣れてくると、彼らはより迅速にプラットフォームを見つけることができます。 1984年にリチャードG.モリスによって開発された、このパラダイムは、行動神経科学の&quot;黄金の基準&quot;の一つとなっている。

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

Morris Water Maze Experiment

モリス水迷路は、行動課題における海馬の関与を文書化するために使用される広く受け入れられているツールです。

モリス水迷路実験

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

56.4K ビュー.  Michigan State University (MSU). モリス水迷路は、行動課題における海馬の関与を文書化するために使用される広く受け入れられているツールです。

ビデオ: 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.

人工飼料の実験を使用してアブラムシにニコチン耐性をテスト

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

生物学

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.

マウス用高架式十字迷路

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 ...

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筋芽細胞を用いた生体エネルギープロファイル実験

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.

土壌中の微生物の残留決定のためのAldononitrile酢酸誘導体化グルコサミンとムラミン酸のGCベースの検出

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.

採用プロテアーゼ及び酸触媒ラベリングワークフロー<sup&gt; 18</sup&gt; O-濃縮水

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.

しわコロニーの開発を使用したバイオフィルム形成を評価するための半定量的アプローチ

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.

新生仔マウス心筋細胞の単離および培養

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.

有害と潜在的に有害な成分の毒性効果を評価するためにハイコンテントスクリーニング分析（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).

マウス表皮ケラチノサイトの単離とインビトロクロノジェニック培養

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

Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals has yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM+ cells were confirmed to express keratin using immunofluorescence microscopy in Krt1-14;mTmG transgenic mice. Human blood samples had 0.18% &#177; 0.0004 EpCAM+ cells (SEM; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% &#177; 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+. In mouse blood, EpCAM+ cells constituted 0.45% &#177; 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates), and in mouse BM, 5.17% &#177; 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, all the EpCAM+ cells were immunoreactive to pan-cytokeratin, as determined by IF microscopy. Results were confirmed using Krt1-14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p &#60; 0.0005) of GFP+ cells present in normal murine BM, that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. We demonstrate a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals.

This paper presents a reproducible method with new findings on the presence of epithelial cells in normal human and mouse blood and bone marrow using flow cytometry and immunofluorescence microscopy. Krt1-14;mTmG transgenic mice were used as an in vivo method to confirm these findings.

EpCAMおよびサイトケラチンが正常なヒトおよびマウスの血液および骨髄において上皮細胞を発現する証拠

Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial ...