JLN was supported by MH 68347.

Setting up the water maze
<ol>
<li>The main component of the water maze set up should be a round pool, about 6 feet in diameter and about 3 feet deep. If you are recording the task with a video camera, make sure all sides of the maze are within the camera&rsquo;s field of view.</li>
<li>Fill up the water maze with tap water, which should be close to 26&deg;C. This may take several hours, so should be done well in advance. Periodically check the water temperature so that it is within one degree of 26&deg;C.</li>
<li>Place the escape platform in the center of the pool. During training, it must be exposed, one inch above the water. This teaches the rat that there is a platform, and that it is the way to get out of the water. Later, after the animal is trained and ready for testing, the escape platform will be just below the surface of the water, and will not be visible because the water will be made opaque with milk or non-toxic paint. &nbsp;&nbsp;&nbsp; Now, the water maze is ready for&nbsp;training the animals.</li>
</ol>
Pre-training for the water maze
<ol>
<li>For the water maze training, the platform should be in the center of the pool and exposed one inch above the surface, so the animal knows that it&rsquo;s there. The water should be within one degree of 26&deg;C.</li>
<li>Each animal will undergo three consecutive trials. First, place the animal on the platform for twenty seconds.</li>
<li>The water maze has 4 starting positions: north, south, east, or west.&nbsp; Take the animal to one of these positions.&nbsp; Lower the animal into the water by supporting it with your hand and bringing it down gently into the water tail-end first. Do not stress the animal out by dunking it in head first.</li>
<li>Let the animal swim/search for the platform for a maximum of 60 seconds. At first, the animal may swim around the edge of the pool looking for a way out.&nbsp; Eventually, the animal will learn to search for the platform and climb up.&nbsp; </li>
<li>Once the rodent reaches the platform, stop the timer, and record the time.&nbsp; If it doesn&rsquo;t find the platform in 60 seconds, then record the time for this trial as one minute. Do not pick up the animal if it fails to reach the platform. Teach the animal that it must swim to the platform. Therefore, gently guide the animal to the platform with your hand.&nbsp; Let the animal sit on the platform for 15 seconds. If it falls or jumps off, gently guide it back. This will train the animal that it must stay on the platform to be rescued from the pool.</li>
<li>Repeat the same procedure for two more trials, starting at a different direction for each trial.</li>
<li>Once the animal has completed all three trials, dry it off with a towel. Repeat the three-trial training process for all the animals consecutively. Keep the directions the same for all of them, and record their times. </li>
<li>Now that the animals are trained, they are ready to perform the water maze test.</li>
</ol>
Water maze testing
<ol>
<li>To begin experimental trials with the water maze, fill up the tank so the platform is one inch below the surface of the water. Use non-fat dry milk, or 125 milliliters of non-toxic white tempera paint, to make the water opaque.&nbsp; </li>
<li>The lighting and water temperature should be the same as in the training process. Each animal will undergo 12 trials, which will be 3 trials&nbsp;for each starting direction. Each trial will last 60 seconds. Before beginning, choose the order of the starting directions. Do not use the same start direction twice in a row, and also do not repeat the same order for any of the directions</li>
<li>Facing the wall of the pool, the animal handler will place the animal in the water, and will then step back from the pool and sit in a designated spot while the animal performs the maze task. An animal behavior tracking system such as the SMART system (San Diego Instruments) can be used to monitor the path, as well as other variables. Videotaping each trial is a less expensive, and perfectly appropriate, alternative.</li>
<li>Monitor the animal until it reaches the platform, and record the time it took. If the animal doesn&rsquo;t reach the platform in 60 seconds, the handler will guide it to the platform, as in training. Either way, let the animal sit for 10 seconds, and then dry it off and return the animal to a holding cage.</li>
<li>Continue with each animal for all 12 trials, with the animal handler returning to the same designated spot during each trial. The order of testing should be: trial 1 for all animals, trial 2 for all animals, trial 3 for all animals, etc. There should be an inter-trial interval of at least 2 minutes. Periodically, clean out the pool, make sure the platform is in place, and check that the water temperature is the same.</li>
<li>After all animals have completed 12 trials, they will each perform one probe trial, in which the platform is removed from the pool. The probe trial is performed to verify the animal&rsquo;s understanding of the platform location, and observe the strategy that the animal follows when it discovers the platform is not there. The handler will release the animal starting from the north. Record the number of times the animal crosses the center of the pool during the&nbsp; 30 seconds.</li>
<li>When all the probe trials are complete, dry off the animals and drain the pool.</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.

The water maze task was development by Morris 5. This task can be altered in numerous ways to investigate working memory, reference memory and task strategy 6. The procedure described here contains two critical variables which represent a deviation from other versions of the water maze: pretraining, and testing on a single day.
Pretraining - the hippocampus is involved in spatial/relational memory 7-8. The water maze specifically tests spatial...

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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

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 non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

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

Morris Water Maze Experiment

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

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 ...

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Biology

56.3K Views.  Michigan State University (MSU). The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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.

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).

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.

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