Le travail a été soutenu par la Fondation suédoise pour la recherche stratégique environnementale (Mistra) à travers le programme DOM et Grant 211684 (BACSIN) du programme-cadre de l&#39;Union européenne Communauté 7e PC. Nous remercions J. Engstrand de l&#39;aide pour filmer l&#39;analyse aux rayons X et L. Tang pour aider avec le récit conceptuel.

1. La culture et la récolte de P. putida <ol><li> Préparer un levain de Pseudomonas putida par inoculation de 100 ml de bouillon de soja tryptique (TSB) avec une colonie de P. putida cellules cultivées sur agar supplémenté avec du bouillon de soja tryptique (TSA). Gardez la culture à 30 ° C sur une planche en secouant fixé à 130 rpm. </li><li> Après 7 h de transférer une fraction aliquote de l&#39;équivalent de levain à 1/10 du volume final de la culture principale pour BST-milieu frais. Gardez les cellules à 30 ° C sur une planche en agitant à 130 rpm pendant 16 heures. </li><li> Après 16 h de croissance, les cellules sont récoltées par centrifugation de la culture cellulaire à 1500 g pendant 20 min à température ambiante. Décanter le surnageant et laver les cellules par remise en suspension du culot cellulaire se trouve dans une solution de NaCl qui est isotonique pour le milieu de croissance. Dans ce cas, une solution de NaCl 150 mM a été utilisé. Les cellules sont ensuite centrifugées de nouveau et le surnageant est décanté avant la remise en suspension des cellules dans le milieu de formulation,voir l&#39;étape 3.2. </li></ol> 2. La culture d&#39;autres espèces <ol><li> Pour adapter le protocole à d&#39;autres espèces bactériennes les procédures de culture et de récolte doivent être modifiées en fonction des besoins de cette espèce. Pour éviter un choc osmotique lors de la manipulation des cellules pendant la récolte et l&#39;étape de lavage (s), l&#39;osmolalité des solutions doit correspondre à celle du milieu de culture à la récolte. </li></ol> 3. Formulation des cellules <ol><li> Préparation des solutions de formulation en pesant les composants de la matrice respective et les dissoudre dans de l&#39;eau. Pour obtenir des conditions isotoniques, soit régler la quantité d&#39;excipient ou ajouter de NaCl ou d&#39;un autre soluté compatible cellulaire pour atteindre l&#39;osmolalité désirée. Pour les substances de faible poids moléculaire comme disaccharides les montants peuvent facilement être adaptées pour atteindre les conditions isotoniques. Pour les substances, comme les polymères qui sont de haut poids moléculaire, la tonicité doit être ajusté avec la cellule compatible siluths, par exemple NaCl ou disaccharides. Notez que toute addition d&#39;une substance va influencer les propriétés physiques de la formulation, ce qui peut à son tour influencer le comportement lyophilisation et la survie cellulaire. </li><li> Décanter la solution de lavage (dans ce cas NaCl) et remettre en suspension les cellules dans les milieux de formulation respectifs. </li><li> Assurez-vous que les cellules sont dispersées de manière homogène. Les formulations de faible viscosité sont vortex alors que les formulations des viscosités plus élevées sont mélangés à l&#39;aide en ajoutant, par exemple., Une barre d&#39;agitation et agitant le récipient jusqu&#39;à ce que le mélange homogène est atteint. </li><li> Répartissez les formulations dans des flacons lyophilisateur. Les flacons doivent être pesés vide, avec l&#39;échantillon avant et après lyophilisation pour calculer la quantité d&#39;eau éliminée lors de la lyophilisation. </li><li> Ajouter des bouchons en caoutchouc pour les flacons si les flacons doivent être scellés à l&#39;intérieur du lyophilisateur, voir l&#39;étape 4.3. </li><li> Énumérer les cellules dans chaque formulation avant la lyophilisation, voir l&#39;étape 6. </li> &lt;/ Ol&gt; 4. La lyophilisation <ol><li> Les conditions de lyophilisation doivent être ajustés aux propriétés physiques de la formulation. Le paramètre le plus important est dans ce cas la température de transition vitreuse, Tg, de la formulation, notée Tg &#39;de l&#39;échantillon de gel-concentré pour indiquer que l&#39;eau est encore présente. Le «Tg de la formulation gel concentré est facilement mesurée par calorimétrie différentielle à balayage (DSC), voir l&#39;étape 7. </li><li> Ajuster les paramètres du procédé de lyophilisation pour que la température de l&#39;échantillon est toujours en dessous de la Tg de l&#39;échantillon et que la pression de la chambre permet une sublimation rapide de la glace, c&#39;est à dire le séchage primaire, et de l&#39;eau résiduelle dans la formulation, à savoir secondaire séchage. Si la température de l&#39;échantillon est au-dessus de Tg »pendant le processus de séchage il ya un grand risque d&#39;effondrement de la structure de l&#39;échantillon qui peut diminuer de façon significative la survie cellulaire. </li><li> Pour protéger les produits secs après freeze-séchage de l&#39;atmosphère de l&#39;échantillon doit être contrôlée. Si possible, sceller les échantillons dans le lyophilisateur avant le vide est libéré à la fin du cycle de lyophilisation. Alternativement, les flacons peuvent être remplis avec une atmosphère préféré. Il est important d&#39;éviter d&#39;exposer les échantillons à des conditions ambiantes comme l&#39;humidité ou d&#39;oxygène présent dans l&#39;atmosphère de stockage va influencer la survie des cellules. </li><li> Peser les flacons. </li></ol> 5. Réhydratation <ol><li> Réhydrater les échantillons avec de l&#39;eau déminéralisée et stérile. La quantité d&#39;eau à ajouter devrait être la même que la quantité prélevée au cours de la lyophilisation et est calculé à partir du poids du flacon vide, le même flacon avec l&#39;échantillon avant et après lyophilisation. Vortex les échantillons de temps en temps alors que les solutions apparaissent homogène. </li></ol> 6. Enumeration <ol><li> Dessinez 100 ul de chaque échantillon et réaliser des dilutions de 10 fois. A partir des dilutions de intereste 100 pi est plaquée sur des plaques de TSA. Les plaques sont incubées à la température et le temps requis. </li></ol> 7. Caractérisation de la formulation de gel Comportement <ol><li> Prenez une petite quantité, 5 à 10 mg, de l&#39;échantillon et placez-le dans une casserole DSC-aluminium avec couvercle. Préparer une casserole vide comme référence. </li><li> Réglez le programme de numérisation de température DSC. Le programme de refroidissement est réglé pour reproduire l&#39;étape de congélation dans le programme de lyophilisation. Ceci est fait comme la cinétique de refroidissement peuvent influencer la «Tg des formulations hydratés. Avant la numérisation de chauffage commence faire baisser la température bien en dessous de la Tg attendue »des formulations étudiées, normalement -100 ° C. </li><li> Choisir un taux approprié de chauffage, normalement une vitesse de chauffage de 5 - 40 ° C / min est utilisé. Le signal de flux thermique enregistrée est exprimée en watts, et est influencé par la vitesse de chauffage. Une vitesse de chauffage plus élevée donnera un signal plus importante de Tg &#39;. </li><li> Définissez la plage de température de sorte que it couvre à la fois «Tg et la fonte de la formulation. </li></ol> 8. Surface mesures de tension des formulations hydratées <ol><li> Le dispositif expérimental utilisé dans cette expérience est conforme à la méthode du Noüy pour mesurer la surface. </li><li> Nettoyer la bague de platine et le récipient de mesure soigneusement. Le navire est rincé avec de l&#39;acétone, essuyé avec un chiffon non pelucheux, puis brûlé à l&#39;intérieur avec une flamme incolore. L&#39;anneau devient rouge dans une flamme incolore. </li><li> Remplissez la cuve avec la solution et laissez la surface régler avant la mesure. Pour les solutions où l&#39;état d&#39;équilibre est atteint seulement après une longue période, par exemple des polymères, la configuration simple utilisé ici fourniront des données qualitatives. </li><li> Pour des formulations ayant des viscosités élevées, par exemple des solutions à 2% (p / p) HEC ou l&#39;HPMC, la tension de surface doit être mesurée sur des solutions de concentrations plus faibles, et ensuite extrapolé. Cela peut être fait en profitant du fait quela baisse des niveaux de tension de surface au large à des concentrations inférieures à 0,5% (p / p). </li></ol> 9. L&#39;analyse aux rayons X des formulations sèches <ol><li> Prendre une petite quantité de matrice / bactéries et le charger sur un porte-échantillon. </li><li> Monter le porte-échantillon dans le X-ray-diffractomètre. </li><li> Pour analyser n&#39;importe quelle phase cristalline présente dans l&#39;échantillon du progiciel EVA de Bruker peut être utilisé. </li></ol> 10. Electron Microscopy <ol><li> Prendre une petite quantité de Matrix / bactéries sur les flacons et le fixer sur un MEB. </li><li> Chargez le support SEM dans la pulvérisation coucheuse (Thermo Polaron VGScientific SC7640 utilisé dans le présent ouvrage) et le manteau de l&#39;échantillon avec quelques nm de Au / Pd ou Pt. Dans ces expériences, les paramètres de pulvérisation étaient: 1,900 V, 20 mA et 20 sec, pour un ~ 5 - 10 nm de revêtement Au / Pd. L&#39;enrobage permet de réduire la charge électrique au cours de l&#39;acquisition d&#39;image. Il est recommandé de commencer avec un revêtement mince et, si la charge persiste et provoque l&#39;imageartefacts, l&#39;échantillon doivent être enduites de nouveau. </li><li> Chargez le support SEM dans la chambre SEM. Sélectionnez les paramètres d&#39;imagerie appropriées. Pour l&#39;instrument utilisé dans la figure 2 (FEI Strata DB235), nous avons utilisé 5 tension d&#39;accélération de kV, taille de mode 3, sur une distance de travail de 5 mm et le détecteur d&#39;électrons secondaires. </li><li> En option, il est possible d&#39;utiliser le faisceau d&#39;ions focalisé pour couper des sections de la bactérie incorporés dans le polymère de révéler plus de détails. </li></ol>

<ol>
	<li>Wessman, P., Mahlin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+matrix+properties+on+the+survival+of+freeze-dried+bacteria.">Impact of matrix properties on the survival of freeze-dried bacteria.</a> J. Sci. Food Agric. 91 (14), 2518-2528 (2011).</li><li>Ablett, S., Izzard, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Scanning+Calorimetric+Study+of+Frozen+Sucrose+and+Glycerol+Solutions.">Differential Scanning Calorimetric Study of Frozen Sucrose and Glycerol Solutions.</a> Journal of the Chemical Society-Faraday Transactions. 88 (6), 789-794 (1992).</li><li>Knopp, S. A., Chongprasert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+type+TMDSC+curve+of+frozen+sucrose+solutions+and+collapse+during+freeze-drying.">The relationship between type TMDSC curve of frozen sucrose solutions and collapse during freeze-drying.</a> Journal of Thermal Analysis and Calorimetry. 54 (2), 659-672 (1998).</li><li>Crowe, J. H., Leslie, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+Vitrification+Sufficient+to+Preserve+Liposomes+during+Freeze-Drying.">Is Vitrification Sufficient to Preserve Liposomes during Freeze-Drying.</a> Cryobiology. 31 (4), 355-366 (1994).</li><li>Jain, P., Sen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+glass-forming+biopreservatives+on+head+group+rotational+dynamics+in+freeze-dried+phospholipid+bilayers:+A+P-31+NMR+study.">Effect of glass-forming biopreservatives on head group rotational dynamics in freeze-dried phospholipid bilayers: A P-31 NMR study.</a> Journal of Chemical Physics. 131 (2), (2009).</li><li>Stock, J. B., Rauch, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periplasmic+Space+in+Salmonella-Typhimurium+and+Escherichia-Coli.">Periplasmic Space in Salmonella-Typhimurium and Escherichia-Coli.</a> Journal of Biological Chemistry. 252 (21), 7850-7861 (1977).</li><li>Crowe, J. H., Hoekstra, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis.">Anhydrobiosis.</a> Annual Review of Physiology. 54, 579-599 (1992).</li><li>Leslie, S. B., Israeli, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+and+Sucrose+Protect+Both+Membranes+and+Proteins+in+Intact+Bacteria+during+Drying.">Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.</a> Applied and Environmental Microbiology. 61 (10), 3592-3597 (1995).</li><li>Nesarikar, V. V., Nassar, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cations+and+anions+on+glass+transition+temperatures+in+excipient+solutions.">Effect of cations and anions on glass transition temperatures in excipient solutions.</a> Pharmaceutical Development and Technology. 12 (3), 259-264 (2007).</li><li>You, Y., Ludescher, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sodium+chloride+on+molecular+mobility+in+amorphous+sucrose+detected+by+phosphorescence+from+the+triplet+probe+erythrosin+B.">The effect of sodium chloride on molecular mobility in amorphous sucrose detected by phosphorescence from the triplet probe erythrosin B.</a> Carbohydr. Res. 343 (2), 350-363 (2008).</li><li>Crowe, J. H., Hoekstra, F. A., Nguyen, K. H. N., Crowe, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+vitrification+involved+in+depression+of+the+phase+transition+temperature+in+dry+phospholipids.">Is vitrification involved in depression of the phase transition temperature in dry phospholipids.</a> Biochim. Biophys. Acta Biomembr. 1280, 187-196 (1996).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Le motif de cette étude était d&#39;étudier certaines propriétés de formulation qui peuvent être d&#39;importance pour la survie des cellules pendant la lyophilisation. Bien que la tolérance intrinsèque de séchage varie selon les espèces, comme illustré à la figure 1A, la tendance de la façon dont les formulations différentes soutien survie cellulaire est similaire. Il est instructif de commencer par une comparaison de saccharose et de Ficoll. On croit qu&#39;un facteur clé pour une formu...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Ficoll PM 400</td>
			<td>GE-healthcare</td>
			<td>17-0300-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEC (Natrosol-M Pharm Grade)</td>
			<td>Ashland</td>
			<td>&nbsp;</td>
			<td>Gift from Ashland</td>
		</tr>
		<tr>
			<td>HPMC (Methocel F4M)</td>
			<td>Dow</td>
			<td>&nbsp;</td>
			<td>Gift from the Department of Pharmacy, Uppsala University</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S2395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptic Soy broth</td>
			<td>Merck</td>
			<td>105459</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>Merck</td>
			<td>105458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lyostar II</td>
			<td>FTS Kinetics</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pyris Diamond DSC</td>
			<td>Perkin-Elmer</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bruker AXS SMART CCD 1k Diffractometer</td>
			<td>Bruker</td>
			<td>N.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-beam FEI Strata DB235 FIB/SEM</td>
			<td>FEI</td>
			<td>N.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kr&uuml;ss Educational Tensiometer</td>
			<td>Kr&uuml;ss</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

formulations for freeze-drying of bacteria and their influence on cell survival

Eau cellulaire peut être retiré pour inactiver les microorganismes réversible pour faciliter le stockage. Une telle méthode de suppression est la lyophilisation, qui est considéré comme une méthode de déshydratation douce. Pour faciliter la survie des cellules pendant le séchage, les cellules sont souvent préparées à l&#39;avance. La formulation forme une matrice qui intègre les cellules et les protège contre divers stress nuisibles imposées sur les cellules lors de la congélation et le séchage. Nous présentons ici une méthode générale pour évaluer le taux de survie des cellules après lyophilisation et nous l&#39;illustrons en comparant les résultats obtenus avec quatre formulations différentes: le saccharose disaccharide, le saccharose dérivé polymère Ficoll PM400, et les polysaccharides respectifs hydroxyéthylcellulose (HEC ) et de l&#39;hydroxypropyl méthyl cellulose (HPMC), sur deux souches de bactéries, de P. putida KT2440 et A. A6 chlorophenolicus. Dans ce travail, nous illustrons comment préparer des formulations pour la lyophilisation et la façon d&#39;enquêter sur la mécaniqueismes de la survie des cellules après réhydratation en caractérisant la formulation en utilisant la calorimétrie différentielle à balayage (DSC), les mesures de tension de surface, analyse aux rayons X et microscopie électronique et concernant ces données à taux de survie. Les polymères ont été choisis de façon à obtenir une structure monomère de la saccharose ressemblant à de polysaccharide correspondant à un des degrés divers. En utilisant cette méthode setup nous avons montré que les polymères peuvent soutenir la survie cellulaire aussi efficacement que disaccharides si certaines propriétés physiques de la formulation sont contrôlées 1.

Le tableau 1 présente les données sur la composition de la formulation, des événements enregistrés par DSC thermiques pendant le chauffage des formulations congelées, de la structure des échantillons secs et de la tension superficielle des solutions de formulation. Le T g &#39;de saccharose a été déterminé à -40 ° C 2, 3 et peut être difficile à détecter pour les concentrations de saccharose inférieure à 20% p / p. L&#39;événement thermique à -35 ° C ...

Watch this Scientific Journal Video about Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival at JoVE.com

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

La lyophilisation est souvent un moyen facile et pratique d&#39;obtenir des produits secs de cellules bactériennes viables. Un problème de ce processus est la survie cellulaire. Nous détaillons ici une procédure pour étudier comment la survie des cellules pendant la lyophilisation est influencée par les propriétés de la formulation utilisée.

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 ...

formulations-for-freeze-drying-bacteria-their-influence-on-cell

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Biology

23.2K vues.  Swedish University of Agricultural Sciences. La lyophilisation est souvent un moyen facile et pratique d'obtenir des produits secs de cellules bactériennes viables. Un problème de ce processus est la survie cellulaire. Nous détaillons ici une procédure pour étudier comment la survie des cellules pendant la lyophilisation est influencée par les propriétés de la formulation utilisée.

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

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

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

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

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

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 ...

Recherche

Biologie

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

Détection des modifications post-traductionnelles sur les autochtones nucléosomes Intact par ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analyse de la fonction des petites GTPases par micro-injection de plasmides dans les cellules épithéliales polarisées

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Profilage Single Cell transcriptionnelle des cardiomyocytes de souris adultes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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 ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Stratégies pour le suivi Anastasis, une cellule de survie Phénomène qui renverse apoptose

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Une méthode standardisée pour l&#39;analyse des cellules endothéliales et sinusoïdale du foie Leurs fenestrations par microscopie électronique à balayage

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Mesure de temps de survie dans<em&gt; Brachionus</em&gt; Rotifères: Synchronisation des conditions maternelles

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Enquête sur les néfastes effets de basse pression Plasma stérilisation sur la survie de Bacillus subtilis Spores en utilisant Live microscopie des cellules

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Quantification des protéines spécifiques au site Lysine acétylation et stoechiométrie Succinylation spectrométrie de masse indépendante des données Acquisition

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...