Les sources de financement: Université du Connecticut Research Foundation et le Département de Biologie Moléculaire et Cellulaire à SVN, Sigma Xi Grant-in-Aid de recherche et Antonio H. et R. Majorie Romano Graduate Education Fellowship pour AJC

<ol><li> Préparer 500 ml de 0,22 um, stérilisée par filtration d&#39;eau de mer artificielle (FSW; salinité 35 ppt). Filtre d&#39;eau de mer artificielle ou naturelle à travers un filtre de 0,22 micron um pour éliminer les particules et les bactéries. </li><li> Anesthetize un adulte Hawaiian bobtail squid (Euprymna scolopes) en le plaçant dans une solution à 2% d&#39;éthanol dans FSW. Placez l&#39;animal dans l&#39;anesthésie pendant environ 10 minutes. Le calmar cessera de natation et ne sera pas répondre activement au toucher. La respiration continue, indiquée par le mouvement du manteau, et l&#39;activité devrait encore chromatophore être observées. </li><li> Placez calmars avec la face ventrale vers le haut sur un plateau de dissection de cire standard. Immerger l&#39;animal avec FSW contenant 2% d&#39;éthanol. </li><li> L&#39;utilisation d&#39;un standard de 200 embouts de pipette ul, tirez l&#39;entonnoir et le manteau pour exposer le vaisseau sanguin principal céphalique située entre les deux yeux. </li><li> En utilisant une seringue de 1 mL stérile avec aiguille de calibre 26,5, de percer le vaisseau sanguin céphalique et de retirer entre 50-100 ul d&#39;hémolymphe. Placez l&#39;hémolymphe dans un tube de 1,5 ml stériles sur la glace. Remarque: Si un animal servira une fois donateurs multiples, ne retirer que 10 à 20 pl d&#39;hémolymphe à un moment donné. Retour de l&#39;animal à un réservoir d&#39;eau de mer normale. L&#39;animal va revivre dans les 30 min. </li><li> Hémocytes fraîchement récoltées sont lavées et remises en suspension dans 500 ul de solution de Ringer Squid s (S-Ringer; 530 mM NaCl, 10 mM de KCl, 25 mM MgCl2, 10 mM de CaCl2 et 10 mM de tampon HEPES, pH 7,5). </li><li> Les concentrations hémocytaires sont déterminées par hématimètre, et environ 2.000 cellules sont ajoutées aux fiches chambrée couvercle en verre, et autorisés à adhérer au verre pendant 10 min à température ambiante. A cette densité, les hémocytes former une monocouche uniforme sur la surface de la lame de verre. </li><li> Pour observer des bactéries se liant à hémocytes hôte, les hémocytes sont exposés à une souche bactérienne marqué par fluorescence tels que Vibrio fischeri ES114 et / ou Vibrio harveyi B392, contenant chacun un journaliste verte fluorescente. V. fischeri ES114 et V. harveyi B392 sont cultivés à la mi-phase log dans une eau de mer tryptone médias (SWT) à 28 ° C dans un agitateur orbital. La densité optique à 600nm est mesurée par spectrophotométrie pour déterminer la densité cellulaire. Les bactéries sont culottées par centrifugation (5000 rpm pendant 5 min), le surnageant est éliminé et le culot est remis en suspension dans S-Ringer. </li><li> 100 000 cellules bactériennes sont ajoutés à chaque chambre ainsi donc qu&#39;il ya 50 bactéries par hémocytes en moyenne. Les hémocytes / mélanges de bactéries sont incubées dans la solution S-Ringer s à 25 ° C pendant 1 h, un temps déterminé pour obtenir le niveau maximum de la liaison. </li><li> Le cytoplasme des hémocytes sont ensuite colorés avec fluorescence CellTracker 0,005% d&#39;Orange (Invitrogen) puis lavé à S-Ringers de visualiser les cellules. </li><li> Hémocytes souillé avec des bactéries associées sont considérées par fluorescence en utilisant soit un Zeiss Découverte V20 stéréoscope fluorescente ou d&#39;un microscope Leica SP2 laser confocale spectrale, et énuméré sur toute la surface de la cellule animale. </li></ol> Les résultats représentatifs Parce que l&#39;hémolymphe céphalopodes contient hémocyanine extracellulaire et non l&#39;hémoglobine, sur l&#39;oxygénation, l&#39;hémolymphe deviendra bleu foncé. Une moyenne de ~ 5000 hémocytes par ul d&#39;hémolymphe seront obtenus en utilisant cette méthode. Après l&#39;adhésion aux lamelles chambrée et une coloration fluorescente, les hémocytes devrait apparaître vives fluorescence rouge et en forme amiboïde. Pour l&#39;adhésion bactérienne, V. fischeri adhèrent mal à la hémocytes (1-2 cellules bactériennes par des cellules sanguines), alors que V. harveyi va adhérer fortement (10-15 cellules bactériennes par hémocytes). <img src="/files/ftp_upload/1714/1714fig1.jpg" alt="Figure 1" /> Figure 1. Adulte Hawaiian bobtail squid Euprymna scolopes montrant la position des vaisseaux sanguins céphalique. <img src="/files/ftp_upload/1714/1714fig2.jpg" alt="Figure 2" /> Figure 2. Résultats de l&#39;exposition aux hémocytes Vibrio fischeri (A) et Vibrio harveyi (B). Rouge, Cell Tracker orange, vert, GFP-étiquetée bactéries.

<ol>
	<li>Koropatnick, T. A., Kimbell, J. R., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+host+hemocytes+during+the+initiation+of+the+squid-Vibrio+symbiosis.">Responses of host hemocytes during the initiation of the squid-Vibrio symbiosis.</a> Biol. Bull. 212, 29-39 (2007).</li><li>McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unseen+forces:+the+influence+of+bacteria+on+animal+development.">Unseen forces: the influence of bacteria on animal development.</a> Dev. Biol. 242, 1-14 (2002).</li><li>McFall-Ngai, M. J., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symbiont+recognition+and+subsequent+morphogenesis+as+early+events+in+an+animal-bacterial+mutualism.">Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism.</a> Science. 254, 1491-1494 (1991).</li><li>Nyholm, S. V., Stewart, J. J., Ruby, E. G., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+between+symbiotic+Vibrio+fischeri+and+the+haemocytes+of+Euprymna+scolopes.">Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes.</a> Environ. Microbiol. 11, 483-493 (2009).</li><li>Nyholm, S. V., McFall-Ngai, M. 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+winnowing:+establishing+the+squid-Vibrio+symbiosis.">The winnowing: establishing the squid-Vibrio symbiosis.</a> Nat. Rev. Microbiol. 2, 632-642 (2004).</li><li>Nyholm, S. V., Stabb, E. V., Ruby, E. G., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+animal-bacterial+association:+recruiting+symbiotic+Vibrios+from+the+environment.">Establishment of an animal-bacterial association: recruiting symbiotic Vibrios from the environment.</a> Proc. Natl. Acad. Sci. USA. 97, 10231-10235 (2000).</li><li>Nyholm, S. V., McFall-Ngai, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+the+light-organ+microenvironment+of+Euprymna+scolopes:+description+of+a+population+of+host+cells+in+association+with+the+bacterial+symbiont+Vibrio+fischeri.">Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri.</a> Biol Bull. 195, 89-97 (1998).</li><li>Visick, K. L., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+fischeri+and+its+host:+it+takes+two+to+tango.">Vibrio fischeri and its host: it takes two to tango.</a> Curr. Opin. Microbiol. 9, 632-638 (2006).</li></ol>

Les études concernant le rôle du système immunitaire dans la médiation de la signalisation moléculaire entre les bactéries bénéfiques et leurs hôtes ont, ces dernières années, apporté une contribution significative à notre compréhension de la co-évolution des eucaryotes avec leur microflore. Le système squid / vibrion a fait ses preuves en tant que système modèle souple pour répondre aux questions fondamentales dans ce domaine 2,3,5,6,8. La lumière de l&#39;organe de scolopes calm...

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<td>Lab-Tek chambered #1.0 borosilicate coverglass system (8-chambers)</td>
<td>&nbsp;</td>
<td>Thermo-Fisher Scientific</td>
<td>155411</td>
<td>&nbsp;</td>
<tr>
<td>26.5 G 1ml latex-free insulin syringe</td>
<td>&nbsp;</td>
<td>Becton, Dickinson and Company</td>
<td>C34551</td>
<td>&nbsp;</td>
<tr>
<td>Cell Tracker Orange</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C34551</td>
<td>&nbsp;</td>
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<td>SteREO Discovery V20 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>SP2 Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
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obtaining hemocytes from the hawaiian bobtail squid euprymna scolopes and observing their adherence to symbiotic and non-symbiotic bacteria

Les études concernant le rôle du système immunitaire dans la médiation de la signalisation moléculaire entre les bactéries bénéfiques et leurs hôtes ont, ces dernières années, apporté une contribution significative à notre compréhension de la co-évolution des eucaryotes avec leur microflore. L&#39;association symbiotique entre les calmars Hawaiian Bobtail,<em&gt; Scolopes Euprymna</em&gt; Et la bactérie bioluminescente<em&gt; Vibrio fischeri</em&gt; A été utilisé comme un système modèle pour comprendre les effets des bactéries bénéfiques sur le développement des animaux. Des études récentes ont montré que les macrophages, comme hémocytes, la seule composante innée cellulaire de l&#39;hôte calmar système immunitaire, jouent probablement un rôle important dans la médiation de l&#39;établissement et le maintien de cette association. Ce protocole va montrer comment obtenir des hémocytes de<em&gt; E. scolopes</em&gt; Et ensuite utiliser ces cellules dans des dosages de liaison bactérienne. Adulte calmars sont d&#39;abord anesthésiés avant d&#39;hémolymphe est recueillie par une seringue par le vaisseau sanguin principal céphalique. Les hémocytes hôte, contenues dans l&#39;hémolymphe extraites, sont respectées lamelles de verre cloisonné et ensuite exposés à la protéine fluorescente verte marquée symbiotique<em&gt; Vibrio fischeri</em&gt; Et non-symbiotique<em&gt; Vibrio harveyi</em&gt;. Les hémocytes sont contre avec un colorant fluorescent (Cell Tracker Orange, Invitrogen), puis visualisé par microscopie fluorescente.

Watch this Scientific Journal Video about Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria at JoVE.com

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Cette vidéo vous montrera comment obtenir des hémocytes (cellules sanguines) de l&#39;Hawaiian calmars bobtail,<em&gt; Scolopes Euprymna</em&gt; Pour une utilisation dans des dosages biologiques et d&#39;adhésion cellulaire bactérienne. Hémocytes seront colorées avec un colorant fluorescent et exposés à la GFP-étiquetée bactéries.

Obtention hémocytes de l&#39;Hawaiian Bobtail Squid Scolopes Euprymna Et en observant leur adhésion aux bactéries symbiotiques et non symbiotiques

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

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Biology

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

13.1K vues.  University of Connecticut. Cette vidéo vous montrera comment obtenir des hémocytes (cellules sanguines) de l'Hawaiian calmars bobtail, Scolopes Euprymna Pour une utilisation dans des dosages biologiques et d'adhésion cellulaire bactérienne. Hémocytes seront colorées avec un colorant fluorescent et exposés à la GFP-étiquetée bactéries.

Vidéo: Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Obtention des oeufs de Xenopus laevis femelles

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Recherche

Biologie

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

Formulations pour la lyophilisation des bactéries et leur influence sur la survie des cellules

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Un protocole simple pour extraire l&#39;hémocytes de chenilles sauvages

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolement et culture des fibres musculaires individuelles et de leurs cellules satellites du muscle squelettique adulte

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Collecte, l&#39;isolement et l&#39;enrichissement des bactéries présentes naturellement magnétotactique de l&#39;Environnement

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

Un procédé d&#39;extraction Pigments de Squid<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Protéine fluorescente verte bibloc pour visualiser les effecteurs livré de bactéries lors de l’Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolement de myocytes auriculaires et ventriculaires murins de haute qualité pour des mesures simultanées des transitoires Ca2+ et du courant calcique de type L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

Établissement de cultures binaires stables de saccharibactéries symbiotiques de la cavité buccale

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Isolement et purification évolutifs de vésicules extracellulaires à partir d’Escherichia coli et d’autres bactéries

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...