Les auteurs tiennent à remercier Nicole M. Nemeth (Université de Géorgie) et Jeanne M. Fair (LANL) pour leur aide dans la récolte des tissus de poulet et Steven A. Springer pour son aide pendant le tournage. La prise en charge de tous les oiseaux de cette étude était en conformité avec les lignes directrices des National Institutes of Health pour l&#39;utilisation des animaux de laboratoire et tous les protocoles ont été approuvés par le soin des animaux institutionnel et comités utilisation à Los Alamos National Security, LLC, opérateur du Los Alamos Laboratoire national au titre du contrat n ° DE-AC52-06NA25396 avec le Département américain de l&#39;Énergie. La prise en charge de la souris dans cette étude est en conformité avec l&#39;UCSD animale protocole approuvé. Tissus humains ont été obtenus dans le cadre du protocole de l&#39;UCSD CISR approuvé. Ce travail a été soutenu par une bourse de 118.645 Université de Californie frais Lab Programme Président (PG) et la subvention NS047101 de l&#39;Institut national des troubles neurologiques et des maladies (laboratoire de microscopie Neuroscience partagée, UC San Diego).

<ol>
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Rep. 12, 319-330 (2010).</li><li>Nochi, T., Kiyono, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+the+mucosal+immune+system.">Innate immunity in the mucosal immune system.</a> Curr. Pharm. Des. 12, 4203-4213 (2006).</li><li>Vareille, M., Kieninger, E., Edwards, M. R., Regamey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+airway+epithelium:+soldier+in+the+fight+against+respiratory+viruses.">The airway epithelium: soldier in the fight against respiratory viruses.</a> Clin. Microbiol. Rev. 24, 210-229 (2011).</li><li>McGuckin, M. A., Linden, S. K., Sutton, P., Florin, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucin+dynamics+and+enteric+pathogens.">Mucin dynamics and enteric pathogens.</a> Nat. Rev. Microbiol. 9, 265-278 (2011).</li><li>Knowles, M. R., Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucus+clearance+as+a+primary+innate+defense+mechanism+for+mammalian+airways.">Mucus clearance as a primary innate defense mechanism for mammalian airways.</a> J. Clin. Invest. 109, 571-577 (2002).</li><li>Johansson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inner+of+the+two+Muc2+mucin-dependent+mucus+layers+in+colon+is+devoid+of+bacteria.">The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 15064-15069 (2008).</li><li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat. Rev. Immunol. 10, 159-169 (2010).</li><li>Hayat, M. A., Hayat, 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=.">.</a> Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy. , 53-70 (2002).</li><li>Cohen, M., Hurtado-Ziola, N., Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABO+blood+group+glycans+modulate+sialic+acid+recognition+on+erythrocytes.">ABO blood group glycans modulate sialic acid recognition on erythrocytes.</a> Blood. 114, 3668-3676 (2009).</li><li>Cohen, M., Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sialome--far+more+than+the+sum+of+its+parts.">The sialome--far more than the sum of its parts.</a> OMICS. 14, 455-464 (2010).</li><li>Ota, H., Katsuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternating+laminated+array+of+two+types+of+mucin+in+the+human+gastric+surface+mucous+later.">Alternating laminated array of two types of mucin in the human gastric surface mucous later.</a> Histochemical J. 24, 86-92 (1992).</li><li>Matsuo, K., Ota, H., Akamatsu, T., Sugiyama, A., Katsuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemistry+of+the+surface+mucous+gel+layer+of+the+human+colon.">Histochemistry of the surface mucous gel layer of the human colon.</a> Gut. 40, 782-789 (1997).</li><li>Boyde, A., Maconnachie, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+lithium+salts+reduces+ethanol+dehydration+shrinkage+of+glutaraldehyde+fixed+tissue.">Treatment with lithium salts reduces ethanol dehydration shrinkage of glutaraldehyde fixed tissue.</a> Histochemistry. 66, 181-187 (1980).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Conservation des épitopes de mucus et de tissus congelés glycane est supérieure à celle des tissus qui ont été incorporées dans de la paraffine. Nous avons démontré la préservation de la couche de mucus sécrété (figures 1 et 3) et de la répartition des structures des glycanes trois (figure 3) dans les tissus congelés par rapport à des tissus inclus en paraffine. Fixateurs spécialisés, comme solution de Carnoy (éthanol à 60%, le chloroforme 30%, 10% d&#39;acide acétique) 17 ont été développés pour une conservation optimale de la couche de mucus dans les échantillons de tissus. De façon optimale, cette solution devrait être utilisé pour recueillir des échantillons de tissus qui se consacrent à des études de mucus et a été montré pour préserver l&#39;aspect lisse de 16-17 couche de mucus. La couche de mucus dans les échantillons congelés non fixées intégré dans un PTOM, semble robuste et dans certaines régions peut se détacher du tissu, mais l&#39;épaisseur de la couche globale est en accord avec celle observée dans les tissus qui ont été fixées avec une solution de Carnoy et embedded dans de la paraffine 16-17. Par exemple, la couche de mucus dans la section du côlon congelé les tissus humains est de ~ 100 um (Figure 1), ce qui est à l&#39;intérieur de la fourchette indiquée pour Carnoy&#39;s fixe échantillon côlon humain 55,4 ± 2,5 microns (plage de 7,7 à 204,8 um) 16. Il a été connu depuis des décennies que les résultats de la déshydratation d&#39;éthanol ~ retrait de 30% des échantillons biologiques 18, et que des solvants organiques tels que le xylène, Citrisolv et des lipides, des glycolipides extrait de chloroforme et, dans une certaine mesure, les protéines provenant des 13 tissus. Le traitement des tissus pour inclusion dans la paraffine comprend les étapes suivantes: fixation (10% du formol tamponné), déshydratation (concentration en éthanol augmente) et de compensation (Citrisolv ou le xylène). En reproduisant les étapes de coupes de tissus congelés non fixées, nous avons montré que des extraits de mucus Citrisolv de coupes de tissus congelés résultant de la morphologie du tissu qui est similaire à celle de tissus inclus dans la paraffine (Figure 2, panneau de droite). En revanche, la couche de mucus n&#39;a pas été modifiée par une incubation avec du formol ou de l&#39;éthanol (Figure 2, gauche et du milieu). Ceci suggère que l&#39;étape de compensation de la procédure de paraffine plongement standard, ce qui nécessite une incubation prolongée dans Citrisolv / xylène, entraîne l&#39;effondrement de la couche de mucus. La fixation au formol ne pas endommager la couche de mucus et surgelés sections de tissus qui ont été fixés à la formaline peut être facilement colorés avec les lectines et les anticorps dirigés contre les glycanes, des glycolipides et des protéines (figures 2, 3 et 6). Ces effets peuvent être négligeables pour l&#39;étude des protéines membranaires et des tissus pathologie, mais ils sont dévastateurs pour les structures fortement hydratées telles que la couche de mucus sécrété. Des études histologiques Cependant de mucines sont encore menées principalement avec des échantillons inclus en paraffine, dont la préservation couche de mucus est sous-optimale. Dans des analyses approfondies de composition de la couche de mucus telles que l&#39;identité précise de soicreted ou glycoprotéine liée à la membrane combinaisons MUC exiger des anticorps spécifiques et la spectrométrie de masse pour l&#39;identification de l&#39;épine dorsale de protéine. Préservation de la couche de mucus n&#39;est que la première exigence pour de telles études. De nombreux laboratoires ont des échantillons de tissus congelés dans l&#39;OCT qui ont été recueillies dans le passé pour des projets différents, ces tissus peuvent être facilement utilisées pour étudier les mucines, des glycolipides et de distribution glycane éliminant la nécessité de recueillir des tissus dans fixateurs spéciaux qui sont conçus uniquement pour la préservation du mucus. Tissus congelés subir une transformation minimale et par conséquent la répartition naturelle des glycanes, qui sont hydratés dans la nature, est conservée. Ceci est particulièrement important dans le domaine des interactions hôte-microbienne. Connaissance de la répartition et de l&#39;abondance naturaliste de mucines sécrétées et les structures des glycanes de nombreux décorer ces molécules &quot;barrière&quot; sera un élément clé dans la compréhension de défense de l&#39;hôte, de l&#39;exploitation et de pathogènes microbiensest.

<table border="1"><tbody><tr><td> Nom du réactif et le matériel </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> 2-méthyl butane </td><td> Fisher Scientific </td><td> 03551-4 </td></tr><tr><td> Kit AEC substrat de la peroxydase </td><td> Vector Labs </td><td> SK-4200 </td></tr><tr><td> Bleu Alcian </td><td> Sigma-Aldrich </td><td> A3157 </td></tr><tr><td> Anti-CA 19-9 anticorps monoclonal </td><td> Calbiochem </td><td> CA1003 </td></tr><tr><td> Anti-MUC5AC anticorps monoclonal </td><td> Millipore </td><td> MAB2011 </td></tr><tr><td> Avidine-biotine blocage kit </td><td> Vector Labs </td><td> SP-2001 </td></tr><tr><td> Biotinylé d&#39;âne anti-anticorps de souris </td><td> Immunoresearch Jackson </td><td> 90863 </td></tr><tr><td> SNA biotinylée </td><td> Vector Labs </td><td> B-1305 </td></tr><tr><td> Albumine de sérum bovinmin </td><td> Sigma-Aldrich </td><td> A4503 </td></tr><tr><td> La chitine-hydrolysat </td><td> Vector Labs </td><td> SP-0090 </td></tr><tr><td> Cryostat-microtome </td><td> Leica Microsystems </td><td> Leica CM 1800 </td></tr><tr><td> Hématoxyline </td><td> Surgipath médicale Ind </td><td> 3801570 </td></tr><tr><td> Peroxyde d&#39;hydrogène à 30% </td><td> Fisher Scientific </td><td> H325-100 </td></tr><tr><td> Jacaline-FITC </td><td> Vector Labs </td><td> FL-1151 </td></tr><tr><td> Mayer hématoxyline </td><td> Sigma-Aldrich </td><td> MHS32 </td></tr><tr><td> Melibiose </td><td> Sigma-Aldrich </td><td> M5500 </td></tr><tr><td> Nuclear Fast Red </td><td> Vector Labs </td><td> H-3403 </td></tr><tr><td> Composé OCT </td><td> VWR International </td><td> 25608-930 </td></tr><tr><td> Streptavidine conjuguée à la peroxydase </td><td> Immunoresearch Jackson </td><td> 94638 </td></tr><tr><td> Réactif de Schiff </td><td> Sciences de la microscopie électronique </td><td> 26052 </td></tr><tr><td> sWGA-Rhodamine </td><td> Vector Labs </td><td> RL1022S </td></tr><tr><td> Anticorps monoclonal TKH2 </td><td> ATCC </td><td> HB-9654 </td></tr><tr><td> VectaMount milieu de montage aqueux </td><td> Vector Labs </td><td> H-5501 </td></tr><tr><td> Cytoseal 60 </td><td> Thermo Scientific </td><td> 8310-4 </td></tr><tr><td> Peel-A-Way moules </td><td> Polysciences Inc </td><td> 18646A-1 </td></tr></tbody></table>

using unfixed, frozen tissues to study natural mucin distribution

Mucines sont complexes et fortement glycosylée O-liés glycoprotéines, qui contiennent plus de 70% en poids de glucides 1-3. Mucines sécrétées, produite par les cellules caliciformes et de la muqueuse gastrique, l&#39;échafaud pour fournir une couche de mucus micromètres d&#39;épaisseur qui tapisse l&#39;épithélium de l&#39;intestin et des voies respiratoires 3,4. En plus des mucines, les couches de mucus également contenir des peptides antimicrobiens, des cytokines, des immunoglobulines et 5-9. La couche de mucus est une partie importante de l&#39;immunité innée de l&#39;hôte, et constitue la première ligne de défense contre les micro-organismes envahisseurs 8,10-12. En tant que tel, le mucus est soumis à de nombreuses interactions avec les microbes, les deux agents pathogènes et de symbiotes, et les mucines sécrétées former une interface importante pour ces interactions. L&#39;étude de ces interactions biologiques implique généralement des méthodes histologiques pour la collecte des tissus et de la coloration. Les deux méthodes les plus couramment utilisés pour la collecte des tissus histologiques et préservanttion dans la clinique et dans les laboratoires de recherche sont: la fixation au formol suivie par inclusion dans la paraffine et la congélation des tissus, puis en intégrant en cryo-protecteurs médias. Échantillons de tissus inclus en paraffine produire des sections avec des qualités optimales pour la visualisation histologique, y compris la clarté et la morphologie bien définie. Cependant, au cours du processus de paraffine intégration d&#39;un certain nombre d&#39;épitopes s&#39;altérer et dans le but d&#39;étudier ces épitopes, des coupes de tissus doivent être traitées ultérieurement de l&#39;une des nombreuses méthodes de démasquage 13. Mucines sécrétées et les lipides sont extraits à partir du tissu au cours de l&#39;étape de compensation de la paraffine enrobage, ce qui nécessite de prolonger l&#39;incubation avec des solvants organiques (xylène ou Citrisolv). Par conséquent, cette approche est sous-optimal pour les études portant sur ​​la nature et la répartition des mucines et de mucus in vivo. En revanche, le gel des tissus à la température de coupe optimale (PTOM) intégrant un supportvides déshydratation et la compensation de l&#39;échantillon, et maintient l&#39;hydratation de l&#39;échantillon. Cela permet une meilleure préservation de la couche de mucus hydraté, et permet ainsi l&#39;étude des nombreux rôles de mucines en biologie épithéliale. Comme cette méthode nécessite un minimum de traitement du tissu, le tissu est conservé dans un état plus naturel. Par conséquent gelés sections tissus ne nécessitent aucun traitement supplémentaire avant la coloration et peut être facilement analysée par des méthodes immunohistochimiques. Nous démontrons la préservation de micromètres d&#39;épaisseur couche de mucus sécrété dans les échantillons congelés du côlon. Cette couche est considérablement réduite lorsque les mêmes tissus sont inclus dans la paraffine. Nous démontrons également immunofluorescence des épitopes présentés sur des glycanes des mucines en utilisant des lectines végétales. L&#39;avantage de cette approche est qu&#39;elle ne nécessite pas l&#39;utilisation de fixateurs spéciaux et permet à partir de tissus congelés qui peuvent déjà être conservés dans le laboratoire.

Watch this Scientific Journal Video about Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution at JoVE.com

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Non fixées échantillons de tissus congelés incorporés dans un milieu de température optimale de coupe (OCT) peut être utilisé pour étudier la distribution de glycosylation naturelle et du mucus sécrété. Dans cette approche de traitement de tissus est minime et la présentation naturelle des glycolipides, des mucines et glycane-épitopes est préservée. Les coupes de tissus peuvent être analysées par immunohistochimie en utilisant la détection par fluorescence ou chromogène.

Utilisation non fixés, des tissus congelés pour étudier la distribution mucine naturel

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by ...

using-unfixed-frozen-tissues-to-study-natural-mucin-distribution

Research

JoVE Journal

Biology

47.5K vues.  University of California, San Diego. Non fixées échantillons de tissus congelés incorporés dans un milieu de température optimale de coupe (OCT) peut être utilisé pour étudier la distribution de glycosylation naturelle et du mucus sécrété. Dans cette approche de traitement de tissus est minime et la présentation naturelle des glycolipides, des mucines et glycane-épitopes est préservée. Les coupes de tissus peuvent être analysées par immunohistochimie en utilisant la détection par fluorescen...

Vidéo: Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated Stimuli modèle animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

En utilisant la réponse optocinétique pour étudier la fonction visuelle du poisson zèbre

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

Inverser approche Morpholino génétique utilisant cardiaque ventriculaire injection à Transfecter tissus difficiles à cibles multiples dans le poisson zèbre Larve

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

À haut débit d&#39;analyse de l&#39;image de Sphéroïdes tumorales: Une application logicielle conviviale pour mesurer la taille de Sphéroïdes automatiquement et avec précision

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Utilisation de la souris des cellules tumorales mammaires pour enseigner Biologie Concepts de base: un module de laboratoire simple

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Utilisation de cellules Caco-2 à étudier Lipid Transport par l&#39;intestin

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

Mucine Agarose Gel Electrophoresis: Western Blot pour les glycoprotéines de haut poids moléculaire,


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address existing knowledge gaps. This protocol represents a demonstrated method for nondestructively sampling rare butterflies for population genetic structure or DNA barcoding analyses. It uses the chorion of hatched butterfly ovae to yield sufficiently high quantity and quality DNA for successful gene sequencing to confirm species identity and quantify genetic variation. It may be particularly useful when other tissue sampling techniques are impractical or unavailable. While developed for a lepidopteran, it nonetheless could easily be adapted for use with other insect species. It was specifically designed with ease of use as a goal to help maximize broad implementation by individuals of varying experience and skill levels, such as community scientists, conservation practitioners, and students, and for use over large geographic areas to facilitate broad population sampling. The data generated can help inform taxonomic and listing decisions, conservation and management actions, and enhance basic ecological research.

Here, we present a straightforward protocol for noninvasive genetic sampling of butterfly populations based on the field collection of residual egg debris. It can be used to confirm species identity and quantify genetic variation. This protocol can be easily adapted to broader groups for community science involvement.

Déployer des scientifiques communautaires pour effectuer des échantillonnages génétiques non destructifs de populations de papillons rares

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address ...

Isolement et profilage de noyaux uniques à partir de tissus de mammifères congelés

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Pleins feux sur l’auteur : Améliorer la découverte de médicaments - Développement de protocoles automatisés et standardisés pour l’extraction de noyaux à partir de tissus congelés 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

Un protocole simple, rapide et partiellement automatisé pour l’isolement de noyaux uniques à partir de tissus de mammifères congelés pour le séquençage d’un seul noyau