Configurando o labirinto aquático <ol><li> O principal componente do labirinto aquático criado deve ser uma piscina redonda, de cerca de 6 metros de diâmetro e cerca de 3 metros de profundidade. Se você estiver gravando a tarefa com uma câmera de vídeo, certifique-se todos os lados do labirinto estão dentro de campo da câmera de vista. </li><li> Preencha o labirinto de água com água da torneira, que deve ser perto de 26 ° C. Isso pode levar várias horas, por isso deve ser feito com bastante antecedência. Verificar periodicamente a temperatura da água de modo que seja dentro de um grau de 26 ° C. </li><li> Coloque a plataforma de fuga no centro da piscina. Durante o treinamento, deve ser exposta, um centímetro acima da água. Isto ensina o rato que há uma plataforma, e que é o caminho para sair da água. Mais tarde, depois que o animal é treinado e pronto para testes, a plataforma de fuga será logo abaixo da superfície da água, e não serão visíveis porque a água será feito opaco com leite ou tinta não tóxica. Agora, o labirinto de água está pronta para o treinamento dos animais. </li></ol> Pré-treinamento para o labirinto de água <ol><li> Para a formação labirinto de água, a plataforma deve estar no centro da piscina e expôs uma polegada acima da superfície, de modo que o animal sabe que ele está lá. A água deve ser dentro de um grau de 26 ° C. </li><li> Cada animal será submetido três ensaios consecutivos. Primeiro, coloque o animal na plataforma de 20 segundo. </li><li> O labirinto de água tem 4 posições de partida: norte, sul, leste ou oeste. Levar o animal a uma dessas posições. Menor o animal na água, apoiando-o com a mão e trazendo-a para baixo suavemente na água rabo-final em primeiro lugar. Não se estresse do animal por dunking-lo de cabeça. </li><li> Deixe a nadar animal / pesquisa para a plataforma para um máximo de 60 segundos. Na primeira, o animal pode nadar em torno da borda da piscina à procura de uma saída. Eventualmente, o animal vai aprender a pesquisa para a plataforma e suba. </li><li> Uma vez que o roedor atinge a plataforma, parar o cronómetro, e registrar o tempo. Se não encontrar a plataforma em 60 segundos, em seguida, registrar o tempo para este julgamento como um minuto. Não pegue o animal se não conseguir alcançar a plataforma. Ensinar o animal que ele deve nadar até a plataforma. Portanto, gentilmente orientar o animal para a plataforma com a mão. Deixe o animal sentar-se na plataforma por 15 segundos. Se ele cai ou pula fora, delicadamente guiá-la de volta. Isso irá treinar o animal que ele deve ficar na plataforma a ser resgatada da piscina. </li><li> Repita o mesmo procedimento para mais dois ensaios, a partir de uma direção diferente para cada julgamento. </li><li> Uma vez que o animal tenha completado todos os três ensaios, seque-o com uma toalha. Repita o processo de formação de três julgamento por todos os animais de forma consecutiva. Mantenha as direções o mesmo para todas elas, e registrar seus tempos. </li><li> Agora que os animais são treinados, eles estão prontos para realizar o teste de labirinto aquático. </li></ol> Testes de labirinto aquático <ol><li> Para começar a ensaios experimentais com o labirinto de água, encher o tanque para que a plataforma é uma polegada abaixo da superfície da água. Não uso leite em pó desnatado, ou 125 mililitros de tinta não tóxica tempera branca, para tornar a água opaca. </li><li> A temperatura de iluminação e de água deve ser o mesmo que no processo de formação. Cada animal será submetido 12 estudos, que serão três ensaios para cada direção de partida. Cada processo irá durar 60 segundos. Antes de começar, escolher a ordem das direções de partida. Não use a direção começar a mesma duas vezes seguidas, e também não repita a mesma ordem para qualquer uma das direções </li><li> Frente para a parede da piscina, o manipulador animal vai colocar o animal na água, e então passo para trás a partir da piscina e se sentar em um local designado, enquanto o animal executa a tarefa de labirinto. Um sistema de rastreamento de comportamento animal, como o sistema SMART (Instruments San Diego) pode ser usado para monitorar o caminho, assim como outras variáveis. Filmando cada ensaio é menos caro, e perfeitamente adequado alternativa. </li><li> Monitorar o animal até atingir a plataforma, e gravar o tempo que levou. Se o animal não alcançar a plataforma em 60 segundos, o handler irá guiá-lo para a plataforma, como em treinamento. De qualquer maneira, deixe o animal sentar-se por 10 segundos, e depois seca-lo e devolver o animal para uma gaiola de exploração. </li><li> Continue com cada animal para todos os 12 ensaios, com o tratador de animais retornando ao mesmo local designado durante cada ensaio. A ordem dos testes devem ser: um teste para todos os animais, ensaio 2 para todos os animais, ensaio de 3 para todos os animais, etc Deve haver um intervalo inter-julgamento de pelo menos 2 minutos. Periodicamente, limpe a piscina, certifique-se a plataforma está em vigor, e verificar que a temperatura da água é a mesma. </li><li> Depois de todos os animais tenham completado 12 testes, cada um irá realizar um ensaio sonda, em which a plataforma é removida do pool. O julgamento da sonda é realizada para verificar a compreensão do animal do local da plataforma, e observar a estratégia que o animal segue quando se descobre a plataforma não está lá. O manipulador irá liberar o animal a partir do norte. Registrar o número de vezes que o animal atravessa o centro da piscina durante os 30 segundos. </li><li> Quando todos os ensaios da sonda são completos, secar os animais e drenar a piscina. </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.

A tarefa de labirinto aquático de Morris foi desenvolvimento pela 5. Esta tarefa pode ser alterada de várias maneiras para investigar a memória de trabalho de memória de referência, ea estratégia a tarefa 6. O procedimento descrito aqui contém duas variáveis ​​críticas que representam um desvio de outras versões do labirinto de água: pré-treinamento e testes em um único dia. Pré-treinamento - o hipocampo está envolvida na memória espacial / relacional 7-8. O labir...

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<td>SMART System</td>
<td>San Diego Instruments</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

O labirinto aquático de Morris é amplamente utilizado para estudar a memória espacial e de aprendizagem. Animais são colocados em uma piscina de água que é colorido opaco com leite desnatado em pó ou tinta não tóxica têmpera, onde devem nadar até uma plataforma escapar escondidos. Porque eles estão na água opaca, os animais não pode ver a plataforma, e não pode contar com cheiro para encontrar a rota de fuga. Em vez disso, eles devem confiar no externo / labirinto extra-pistas. Como os animais se tornam mais familiarizados com a tarefa, eles são capazes de encontrar a plataforma mais rapidamente. Desenvolvido por Richard G. Morris, em 1984, este paradigma tornou-se um dos &quot;padrões ouro&quot; de neurociência comportamental.

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

Morris Water Maze Experiment

O labirinto aquático de Morris é uma ferramenta bem aceita utilizado para documentar o envolvimento do hipocampo em uma tarefa comportamental.

Morris Experiment Maze Água

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 visualizações.  Michigan State University (MSU). O labirinto aquático de Morris é uma ferramenta bem aceita utilizado para documentar o envolvimento do hipocampo em uma tarefa comportamental.

Vídeo: 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.

Teste de tolerância à nicotina em Aphids Usando uma experiência Dieta Artificial

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

Biologia

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.

Labirinto elevado para Ratos

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.

Técnicas de imagem Ca 2 + Sinalização no esperma humano

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.

Experimento Perfil bioenergética usando células C2C12 mioblastos

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.

GC baseado em detecção de ácido Aldononitrile Glucosamina e Murâmicos Acetato derivatizados à determinação do resíduo microbiana em solo

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.

Protease e catalisada por ácido Workflows Labeling Empregando<sup&gt; 18</sup&gt; O-enriquecido Água

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.

Uma abordagem semi-quantitativa para avaliar formação de biofilmes usando o desenvolvimento da colônia enrugado

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.

Isolamento e Cultura de Neonatais rato Cardiomyocytes

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.

Análise alto teor de triagem para avaliar os efeitos toxicológicos de Constituintes nocivas e potencialmente prejudiciais (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).

Isolamento dos keratinócitos epidérmicos do camundongo e sua cultura Clonogênica in vitro

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