Financiamento fornecido pelo NIH (1 K08 DK080221-01) e fundos de desenvolvimento institucional Vanderbilt.

Parte 1: Lesão Modelo de Colite <ol><li> Peso inicial deve ser obtido para cada rato antes do início do tratamento DSS. </li><li> Fazer uma de 3% (w / v) Sulfato de Dextran solução de sal de sódio em água e filtrar com um filtro de 0,45 mM de acetato de celulose. </li><li> Substituir a água potável na gaiola do rato com a solução DSS 3% por quatro dias. Os ratos não devem ter acesso a qualquer outra fonte de água (ou seja, a exclusão dicas colocados em sistemas de rega automática). </li><li> No dia quatro, substitua a solução DSS com água por mais três dias, permitindo alguma recuperação do cólon epiteliais. Os ratos devem ser pesados ​​no dia quatro, a fim de quantificar conseqüências sistêmicas da colite. Perda de peso é comum com ferimentos graves. </li><li> No dia 7, pesar e sacrifício dos ratos. Ratos podem ser sacrificados por overdose inalatória de isoflurano, ou outros institucionalmente, IACUC métodos aprovados. </li></ol> Parte 2: Necropsia e Colheita de Colon <ol><li> Expor o lado ventral do rato e pernas seguro para garantir o acesso livre para o abdômen. Molhar o abdômen completamente com álcool 70%. </li><li> Segure a linha média do abdómen com uma pinça e estender para cima, assim tenting a pele. Use uma tesoura de ponta fina para o abdômen inciso expondo o conteúdo abdominal. Estender a incisão para a ponta do apêndice xifóide na linha média e depois estender ao longo do aspecto inferior das margens costal, bilateralmente. </li><li> Identificar o intestino delgado, ceco e cólon. Dissecar cuidadosamente / provocação do cólon a partir do mesentério circundante. Transecto do cólon profundamente na pelve, para libertar o cólon distal e reto. Transecto os dois pontos na margem colonocecal para libertar o cólon proximal. Cuidados devem ser tomados durante este processo para não danificar o colon como a limpeza do cólon será problemático se ocorrer perfuração. </li><li> Utilizando uma agulha 20G alimentação e seringa de 10 ml, entubar e lave o cólon com gelado PBS até que o eluido está completamente limpa de fezes. </li><li> Neste ponto, se a análise macroscópica com um microscópio de dissecação é desejado, os dois pontos podem ser fixados como &quot;lingüiça&quot;. Se a análise histológica é apenas desejável do que prosseguir para a Parte 4 &quot;Fazendo Rolls Suíça para a análise histológica de colite aguda&quot;. </li></ol> Parte 3: Processamento como &quot;Lingüiça&quot; para análise macroscópica de todo o cólon <ol><li> É importante manter a correta orientação do cólon, portanto, manter a extremidade distal do cólon mais próximo do operador. Corte dois pedaços de sutura não absorvível (SP116 1,5 seda trançada métricas), uma polegada de aproximadamente 1 em comprimento, os outros 2 polegadas. </li><li> Use a peça 2 polegada de sutura para amarrar a extremidade distal mais próximo à margem quanto possível, mantendo uma boa vedação. </li><li> Coloque uma agulha 20G alimentação contendo 5 ml de tampão fosfato 10% de formol na extremidade proximal do cólon. Vagamente empate a parte restante da sutura imediatamente proximal ao bulbo da agulha de alimentação. Segure firmemente os dois pontos no bulbo da agulha de alimentação e infundir formalina até o cólon é expandido. Apertar o nó na sutura que você retire a agulha de alimentação, deixando a infusão de cólon, expandiu como um &quot;salsicha&quot;. </li><li> Encha um tubo de 15 ml com 10% de fosfato tamponada de formalina e coloque os dois pontos no tubo. Correção para 24 horas. </li><li> Deitar fora formalina e substituir com EtOH 70% para um 24 horas adicionais. (Colons podem ser armazenados em EtOH 70% indefinidamente à temperatura ambiente.) </li><li> Remova os dois pontos do tubo cônico e cortou as cordas em cada extremidade com um bisturi, tomando cuidado para não danificar o colon. Lembre-se que a extremidade distal tem o maior pedaço de corda. Corte longitudinal do distal para extremidade proximal do cólon para que forme uma longa folha. Neste ponto, o cólon pode ser visto sob um microscópio de dissecação. </li></ol> Parte 4: Making Rolls Suíça para a análise histológica de colite aguda <ol><li> É importante manter a correta orientação do cólon, portanto, manter a extremidade distal do cólon mais próximo do operador. </li><li> Meça o comprimento do cólon. Esta métrica é um indicador da gravidade da lesão. Colite aumenta edema e encurta o comprimento do cólon em geral. </li><li> Usando uma tesoura de ponta fina, inciso longitudinalmente de distal para proximal final do cólon. Use uma pinça bem cotado para agarrar a borda da incisão e aberto lateralmente trabalhando o seu caminho distalmente-&gt; proximalmente, assim, mostrando o cólon como uma folha plana. </li><li> Rolando o cólon exige um par de fórceps e uma técnica de duas mãos. Segure a borda da margem distal com uma pinça. Proceder à seqüencialmente rolo do cólon através da rotação e liberando a pinça de prosseguir para a margem proximal. Gerando assim uma espiral com uma terceira dimensão ou &quot;Swiss roll&quot;.Para manter a forma rolada, segure o rolo com firmeza com uma pinça e transectos com uma agulha 27G1 / 2. Segura a agulha, dobrando a agulha que sai do rolo. </li><li> Coloque o rolo em uma fita de tecido devidamente rotulados e coloque em um frasco de 10% de fosfato tamponada de formol em temperatura ambiente por 24 horas para garantir a fixação do tecido. </li><li> Deitar fora formalina e substituir com EtOH 70% para um 24 horas adicionais. (Colons podem ser armazenados em EtOH 70% indefinidamente à temperatura ambiente.) </li><li> Uma vez que a amostra foi corrigido, ele pode ser incluído em parafina, seccionado e montado em lâminas para análise histológica. </li></ol> Parte 5: Resultados Representante O modelo DSS para a colite aguda permite ao pesquisador obter, corrigir e analisar dois pontos que a colite aguda modelos. Quando o rolo suíço é cortado e montado, ele deve formar uma fatia representativa de todo o cólon, se rolou corretamente (Figura 1). O rolo montado podem ser marcadas com H &amp; E, a fim de determinar a extensão dos danos para o cólon, desde o final (para dentro) distal até o final (fora) proximal (Figura 2). Imunohistoquímica também pode ser realizada no rolo suíço para identificar e quantificar infiltrados inflamatórios. Se o método de lingüiça foi realizado corretamente, os dois pontos fixos será dilatada e toda a superfície da mucosa pode ser facilmente manipulado e visto sob o microscópio de dissecação (Figura 3). <a href="https://www.jove.com/files/ftp_upload/1652/1652fig1.jpg"><img src="/files/ftp_upload/1652/1652fig1tmb.jpg" alt="Figura 1" border="0" /></a> Figura 1. Corretamente executados &quot;Swiss roll&quot;. (A) Os dois pontos são rolados do distal para proximal final, seccionado com uma agulha e garantidos por dobrar a ponta da agulha. Em seguida, é colocada em um cassete de tecido para fixação. (B) H &amp; E manchada seção 5 mM de um rocambole feito a partir do cólon de um rato tratados com DSS (d = cólon distal p = cólon proximal). <a href="https://www.jove.com/files/ftp_upload/1652/1652fig2.jpg"><img src="/files/ftp_upload/1652/1652fig2tmb.jpg" alt="Figura 2" border="0" /></a> Figura 2. Colons DSS tratados mostrar sinais de colite aguda. Inflamação e danos cripta são aparentes no cólon DSS-tratados em comparação com um controle de água tratada. <a href="https://www.jove.com/files/ftp_upload/1652/1652fig3.jpg"><img src="/files/ftp_upload/1652/1652fig3tmb.jpg" alt="Figura 3" border="0" /></a> Figura 3. Um exemplo de uma &quot;salsicha&quot;. A salsicha infundido com formalina e completamente expandidas. Um leve ângulo estará presente secundária à curvatura natural do cólon. A salsicha aberta deve estar nivelado.

<ol>
	<li>Okayasu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98 (3), 694-702 (1990).</li><li>Martinez, 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=Deletion+of+Mtgr1+sensitizes+the+colonic+epithelium+to+dextran+sodium+sulfate-induced+colitis.">Deletion of Mtgr1 sensitizes the colonic epithelium to dextran sodium sulfate-induced colitis.</a> Gastroenterology. 131 (2), 579-588 (2006).</li><li>Moolenbeek, C., Ruitenberg, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Swiss+roll":+a+simple+technique+for+histological+studies+of+the+rodent+intestine.">The "Swiss roll": a simple technique for histological studies of the rodent intestine.</a> Lab Anim. 15 (1), 57-59 (1981).</li><li>Mahler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis.">Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis.</a> Am J Physiol. 274, G544-551 (1998).</li><li>Aharoni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+therapeutic+effect+of+glatiramer+acetate+on+several+murine+models+of+inflammatory+bowel+disease.">Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease.</a> J Pharmacol Exp Ther. 318 (1), 68-78 (2006).</li><li>Hahm, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+transforming+growth+factor+beta+signalling+in+the+intestine+contributes+to+tissue+injury+in+inflammatory+bowel+disease.">Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease.</a> Gut. 49 (2), 190-198 (2001).</li><li>Green, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+modulation+of+Escherichia+coli+O157:H7+adherence+to+the+colonic+mucosa.">Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa.</a> Am J Physiol Gastrointest Liver Physiol. 287 (6), G1238-1246 (2004).</li><li>Dieleman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+experimental+colitis+induced+by+dextran+sulphate+sodium+(DSS)+is+characterized+by+Th1+and+Th2+cytokines.">Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.</a> Clin Exp Immunol. 114 (3), 385-391 (1998).</li></ol>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pela Vanderbilt IACUC.

Este protocolo pode ser modificado para modelo de lesão aguda, lesão de reparação, ou crônica processos de lesão do cólon. Modificação lesão aguda: DSS ad lib tratamento para 4-6 dias, seguido pelo sacrifício imediato Lesão-Modificação: Lesões com 4-6 dias de tratamento DSS seguido por período de recuperação de 3-4 dias e sacrifício (como descrito no protocolo acima). ...

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		<th>Catalogue Number</th>
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<tr>
<td>Dextran Sulfate Sodium Salt</td>
<td>&nbsp;</td>
<td>USB Corp</td>
<td>9011-18-1</td>
<td>mol wt 40,000-50,000</td>
</tr>
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<td>10% Buffered Formalin Phosphate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>SF100-4</td>
<td>&nbsp;</td>
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<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Phoenix Pharmaceuticals</td>
<td>NDC 57319-474-06</td>
<td>&nbsp;</td>
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<td>TISSUE PATH Macrosette Cassettes</td>
<td>&nbsp;</td>
<td>Fisher Healthcare</td>
<td>15182706</td>
<td>&nbsp;</td>
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<td>BD 10 ml Syringe</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>309604</td>
<td>&nbsp;</td>
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<td>Ethyl Alcohol</td>
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<td>Pharmco-Aaper</td>
<td>E190</td>
<td>Dilute to 70% with distilled water</td>
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<td>20G Straight Feeding Needle</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20068-612</td>
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<td>27G1/2 PrecisionGlide Needle</td>
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<td>82027-588</td>
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<td>Waugh Forceps</td>
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<td>&nbsp;</td>
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<td>Non-absorbable Suture </td>
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<td>LOOK Surgical Specialties</td>
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<td>&nbsp;</td>
<td>VWR</td>
<td>28298-020</td>
<td>Cut as needed</td>
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<td>Cole-Parmer</td>
<td>EW-06731-2</td>
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<td>Carbon fiber composites digital caliper</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-007-958</td>
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murine colitis modeling using dextran sulfate sodium (dss)

Colite pode ocorrer por infecções virais ou bacterianas, insulto isquêmico, ou doenças auto-imunes; Colite Ulcerativa e mais notadamente a variante do cólon da doença de Crohn - Colite de Crohn. Colite aguda podem apresentar dor abdominal e distensão, má absorção, diarréia, hematoquezia e muco nas fezes. Estamos começando a entender as complexas interações entre o meio ambiente, genética e disfunção da barreira epitelial em modelos de doença inflamatória intestinal e animal de colite têm sido essenciais para fazer avançar a nossa compreensão da doença. Um modelo popular envolve completando a água potável de camundongos com sulfato de baixo peso molecular de sódio Dextran (DSS), resultando em danos epiteliais e uma resposta robusta inflamatória no cólon de vários dias<sup&gt; 1</sup&gt;. Variações dessa abordagem pode ser usada para modelo de lesão aguda, lesão aguda seguida de reparo, e ciclos repetidos de DSS intercaladas com recuperação de modelagem doenças inflamatórias crônicas<sup&gt; 2</sup&gt;. Após um tratamento de quatro dias de DSS 3% em água potável, os camundongos mostram sinais de colite aguda, incluindo a perda de peso, sangue nas fezes e diarréia. Os ratos são sacrificados na conclusão do curso de tratamento e em dois pontos necropsia dissecados são processados ​​e pode ser &quot;enrolado suíço&quot;<sup&gt; 3</sup&gt; Para permitir a análise microscópica de todo o cólon ou infundido com formol como &quot;lingüiça&quot; para permitir a análise macroscópica. Tecido é então embebidos em parafina, seccionados e corados para análise histológica.

Watch this Scientific Journal Video about Murine Colitis Modeling using Dextran Sulfate Sodium DSS at JoVE.com

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfato de sódio (DSS), administrada na água de beber é um modelo de lesão inflamatória estabelecida murino de colite aguda. Este protocolo descreve o método para o tratamento DSS ea preparação dos tecidos.

Modelagem Colite murino utilizando Dextran Sulfato de Sódio (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

murine-colitis-modeling-using-dextran-sulfate-sodium-dss

Research

JoVE Journal

Biology

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

49.7K visualizações.  Vanderbilt University. Dextran sulfato de sódio (DSS), administrada na água de beber é um modelo de lesão inflamatória estabelecida murino de colite aguda. Este protocolo descreve o método para o tratamento DSS ea preparação dos tecidos.

Vídeo: Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Prever a eficácia da estratégia de substituição População Usando Modelagem Matemática

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

Biologia

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Rotulagem de linhagem de células Zebrafish com Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Investigando inflamação intestinal em DSS-induzida Modelo de IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimizado coloração e Proliferação Métodos de Modelagem para Monitoramento Divisão Celular usando Corantes Rastreamento celulares

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Avaliando Murino Resistência Função Usando Miografia Pressão Arterial

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modelagem do Morbid Genoma Humano utilizando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Imagem latente FITC-dextrano como repórter por exocitose regulada

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

In situ Exploração de Murine Megacaryopoiesis usando Microscopia eletrônica de transmissão

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Avaliação da integridade da barreira intestinal em camundongos usando Dextrano marcado com fluoresceína-isotiocianato

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

Simulando e analisando bicamadas lipídicas usando dinâmica molecular

Realistic Membrane Modeling Using Complex Lipid Mixtures in Simulation Studies

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different mechanical and structural properties in the membrane that directly impact the molecules and processes that occur at this interface. Lipid composition is dynamic and can serve to modulate cell signaling processes. Computational approaches are increasingly used to predict interactions between biomolecules and provide molecular insights to experimental observables. Molecular dynamics (MD) is a technique based on statistical mechanics that predicts the movement of atoms based on the forces that act on them. MD simulations can be used to characterize the interaction of biomolecules. Here, we briefly introduce the technique, outline practical steps for beginners who are interested in simulating lipid bilayers, demonstrate the protocol with beginner-friendly software, and discuss alternatives, challenges, and important considerations of the process. Particularly, we emphasize the relevance of using complex lipid mixtures to model a cell membrane of interest to capture the appropriate hydrophobic and mechanical environments in simulation. We also discuss some examples where membrane composition and properties modulate the interactions of bilayers with other biomolecules.

Autor em destaque: Avançando a biofísica da membrana celular - explorando interações e desafios por meio de abordagens experimentais e computacionais 

Membrane lipid diversity in structure and composition is an important contributor to cellular processes and can be a marker of disease. Molecular dynamics simulations allow us to study membranes and their interactions with biomolecules at atomistic resolution. Here, we provide a protocol to build, run, and analyze complex membrane systems.

Modelagem Realística de Membranas Utilizando Misturas Lipídicas Complexas em Estudos de Simulação

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different ...