Il lavoro è stato sostenuto dalla Fondazione Svedese per la Ricerca Ambientale Strategica (MISTRA) attraverso il programma di DOM e Grant 211684 (BACSIN) dal programma quadro della Comunità europea del 7 ° PQ. Ringraziamo J. Engstrand per l&#39;assistenza a filmare l&#39;analisi a raggi X e L. Tang per aiutare con la narrazione concettuale.

1. Coltivazione e raccolta di P. putida <ol><li> Preparare una coltura di Pseudomonas putida inoculando 100 ml di brodo di soia trittico (TSB) con una colonia di P. cellule putida coltivati ​​su agar addizionato con trittico brodo di soia (TSA). Mantenere la coltura a 30 ° C su una tavola oscillante impostata a 130 rpm. </li><li> Dopo 7 ore trasferire un&#39;aliquota dal coltura starter equivalente a 1/10 del volume finale della coltura principale per fresco TSB-medium. Mantenere le cellule a 30 ° C su un bordo di agitazione a 130 rpm per 16 ore. </li><li> Dopo 16 ore di crescita le cellule vengono raccolte a girare la coltura cellulare a 1500 xg per 20 minuti a temperatura ambiente. Decantare il surnatante e lavare le cellule da ri-sospende il pellet cellulare è in una soluzione di NaCl che è isotonica al mezzo di crescita. In questo caso è stata utilizzata una soluzione 150 mM di NaCl. Le cellule vengono quindi centrifugate nuovamente e il supernatante viene decantato prima di risospendere le cellule nel mezzo formulazione,vedere il punto 3.2. </li></ol> 2. La coltivazione di altre specie <ol><li> Per adattare il protocollo ad altre specie batteriche le procedure di coltivazione e di raccolta dovrebbero essere modificati in base alle esigenze di quella specie. Per evitare shock osmotico durante la manipolazione delle cellule durante la vendemmia e la fase di lavaggio (s), l&#39;osmolalità delle soluzioni deve corrispondere a quella del mezzo di crescita al momento della raccolta. </li></ol> 3. Formulazione di cellule <ol><li> Preparare le soluzioni di formulazione pesando i rispettivi componenti della matrice e si sciolgono in acqua. Di ottenere condizioni isotoniche, sia regolare la quantità di eccipiente o aggiungere NaCl o qualche altro soluto compatibile cella per raggiungere la osmolalità desiderato. Per le sostanze a basso peso molecolare, come disaccaridi gli importi possono essere facilmente regolati per raggiungere le condizioni isotoniche. Per sostanze come polimeri che sono di alto peso molecolare, la tonicità deve essere regolata con la cellula in modo compatibileliuti, ad esempio NaCl o disaccaridi. Si noti che l&#39;eventuale aggiunta di una sostanza influenza le proprietà fisiche della formulazione, che a loro volta possono influenzare il comportamento liofilizzazione e la sopravvivenza cellulare. </li><li> Decantare la soluzione di lavaggio (in questo caso NaCl) e risospendere le cellule in rispettivi mezzi di formulazione. </li><li> Assicurarsi che le cellule sono omogeneamente dispersi. Formulazioni di bassa viscosità sono in agitazione mentre formulazioni di viscosità più elevate sono mescolati con l&#39;aggiunta, per esempio., Un ancoretta e scuotendo il contenitore fino a miscelazione omogenea si ottiene. </li><li> Dividete le formulazioni in fiale liofilizzatore. I flaconcini devono essere pesate vuote, con il campione prima e dopo liofilizzazione per calcolare la quantità di acqua rimossa durante la liofilizzazione. </li><li> Aggiungi ai tappi di gomma dei flaconcini se le fiale devono essere sigillate all&#39;interno del liofilizzatore, vedere il punto 4.3. </li><li> Enumerare le cellule in ogni formulazione prima della liofilizzazione, vedi punto 6. </li> &lt;/ Ol&gt; 4. Liofilizzazione <ol><li> Le condizioni di liofilizzazione devono essere adattati alle proprietà fisiche della formulazione. Il parametro più importante è in questo caso la temperatura di transizione vetrosa, Tg, della formulazione, denotato Tg &#39;per il campione freeze-concentrato per indicare che l&#39;acqua è ancora presente. Il Tg &#39;della formulazione freeze-concentrato è facilmente misurata con la calorimetria differenziale a scansione (DSC), vedere punto 7. </li><li> Regolare i parametri del processo di liofilizzazione modo che la temperatura del campione è sempre inferiore alla Tg del campione e che la pressione in camera consente un rapido sublimazione del ghiaccio, cioè essiccazione primarie, e l&#39;acqua residua nella formulazione, cioè secondaria essiccazione. Se la temperatura del campione è superiore Tg &#39;durante il processo di essiccazione è un grande rischio di collasso strutturale del campione che può diminuire significativamente la sopravvivenza cellulare. </li><li> Per proteggere i prodotti secchi dopo freeze-essiccazione l&#39;atmosfera campione deve essere controllato. Se possibile, sigillare i campioni nel liofilizzatore prima che il vuoto viene rilasciato alla fine del ciclo di liofilizzazione. In alternativa, i flaconi possono essere riempiti con un&#39;atmosfera preferito. È importante evitare di esporre i campioni alle condizioni ambientali di umidità o ossigeno presente nell&#39;atmosfera stoccaggio influenzerà la sopravvivenza delle cellule. </li><li> Pesare le fiale. </li></ol> 5. Reidratazione <ol><li> Reidratare i campioni con acqua deionizzata e sterile. La quantità di acqua da aggiungere che dovrebbe essere uguale alla quantità rimossa durante la liofilizzazione e viene calcolato dal peso del flacone vuoto, lo stesso flaconcino con campione prima e dopo la liofilizzazione. Vortex i campioni di oggi e di nuovo poi fino a quando le soluzioni appaiono omogenee. </li></ol> 6. Enumerazione <ol><li> Disegna 100 microlitri di ciascun campione e fare diluizioni seriali di 10 volte. Dalle diluizioni di interiposo 100 microlitri è placcato su TSA-piastre. Le piastre vengono incubate a temperatura e tempo richiesto. </li></ol> 7. Caratterizzazione di Formulazione di comportamento di blocco <ol><li> Prendete una piccola quantità, 5 - 10 mg, del campione e racchiuderlo in una padella DSC-alluminio con coperchio. Preparare una pentola vuota come riferimento. </li><li> Impostare il programma di scansione di temperatura DSC. Il programma di raffreddamento è impostato per imitare il passo congelamento nel programma liofilizzazione. Questo viene fatto come la cinetica di raffreddamento possono influenzare la Tg &#39;delle formulazioni idrati. Prima della scansione riscaldamento inizia abbattere la temperatura al di sotto della Tg atteso &#39;delle formulazioni indagate, normalmente -100 ° C. </li><li> Scegliere un tasso adeguato riscaldamento, normalmente una velocità di riscaldamento di 5 - 40 ° C è utilizzato / min. Il segnale di flusso di calore registrata è espresso in watt e verrà influenzata dalla velocità di riscaldamento. Un tasso di riscaldamento più alto darà un segnale maggiore di Tg &#39;. </li><li> Impostare l&#39;intervallo di temperatura in modo che it copre sia Tg &#39;e lo scioglimento della formulazione. </li></ol> 8. Tensione superficiale Misure delle formulazioni idrate <ol><li> Il setup sperimentale utilizzato in questo esperimento è secondo il metodo per misurare du Noüy superficie. </li><li> Pulire l&#39;anello di platino e il vaso di misura con attenzione. Il vaso viene risciacquata con acetone, si asciugò con un panno privo di pelucchi, e poi bruciato all&#39;interno con una fiamma incolore. L&#39;anello si illumina di rosso in una fiamma incolore. </li><li> Riempire il serbatoio con la soluzione e lasciare che la superficie di risolvere prima di misurare. Per le soluzioni in cui si raggiunge lo stato di equilibrio solo dopo un lungo periodo di tempo, ad esempio, i polimeri, la semplice impostazione adottata permette di fornire dati qualitativi. </li><li> Per le formulazioni con alte viscosità, ad esempio miscele di 2% (w / w) o HEC HPMC, la tensione superficiale deve essere misurata su soluzioni a concentrazioni inferiori e poi estrapolato. Questo può essere fatto sfruttando il fatto chela caduta dei livelli di tensione superficiale off in concentrazioni inferiori a 0,5% (w / w). </li></ol> 9. X-ray analisi delle formulazioni a secco <ol><li> Prendere una piccola quantità di matrice / batteri e caricarla su un supporto del campione. </li><li> Montare il supporto del campione nella radiografia-diffrattometro. </li><li> Per analizzare qualsiasi fase cristallina presente nel campione pacchetto del programma EVA da Bruker può essere utilizzato. </li></ol> 10. Microscopia Elettronica <ol><li> Prendere una piccola quantità di matrice / batteri fuori delle fiale e fissarlo su uno stub SEM. </li><li> Caricare il supporto di SEM in sputtering-verniciatore (Thermo Polaron VGScientific SC7640 utilizzato nel presente lavoro) e coprire il campione con pochi nm di Au / Pd o Pt. In questi esperimenti i parametri di sputtering sono stati: 1.900 V, 20 mA, e 20 sec, per un ~ 5-10 nm rivestimento Au / Pd. Il rivestimento si riduce la carica elettrica durante l&#39;acquisizione dell&#39;immagine. Si consiglia di iniziare con un rivestimento sottile e, se la carica persiste e provoca immagineartefatti, il campione devono essere rivestite di nuovo. </li><li> Caricare il supporto SEM nella camera SEM. Selezionare i parametri di imaging appropriate. Per lo strumento utilizzato nella Figura 2 (FEI Strata DB235) abbiamo usato 5 kV tensione di accelerazione, spotsize 3, una distanza di lavoro di 5 mm e il rivelatore di elettroni secondari. </li><li> Facoltativamente, è possibile utilizzare il Focused Ion Beam per tagliare le sezioni trasversali dei batteri incorporati nel polimero per rivelare ulteriori dettagli. </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>

Nessun conflitto di interessi dichiarati.

Il motivo di questo studio era di studiare alcune proprietà di formulazione che possono essere di importanza per la sopravvivenza delle cellule durante la liofilizzazione. Sebbene la tolleranza asciugatura intrinseco varia tra specie diverse, come illustrato nella Figura 1A, la tendenza da quanto la formulazioni differente supporto sopravvivenza cellulare è simile. È istruttivo per iniziare con un confronto di saccarosio e di Ficoll. Si ritiene che un fattore chiave per una formulazione per supportar...

<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

Acqua cellulare può essere rimosso per inattivare microrganismi reversibilmente esigenze di stoccaggio. Uno di questi metodi di rimozione è liofilizzazione, che è considerato un metodo di disidratazione delicato. Per facilitare la sopravvivenza cellulare durante l&#39;asciugatura, le cellule sono spesso formulate in anticipo. La formulazione forma una matrice che incorpora le cellule e le protegge da varie sollecitazioni dannose imposte alle cellule durante il congelamento e l&#39;essiccazione. Presentiamo qui un metodo generale per valutare il tasso di sopravvivenza delle cellule dopo liofilizzazione e illustriamo lo confrontando i risultati ottenuti con quattro diverse formulazioni: disaccaride saccarosio, il saccarosio derivato polimero Ficoll PM400, e le rispettive polisaccaridi idrossietilcellulosa (HEC ) e idrossipropilmetilcellulosa (HPMC), su due ceppi di batteri, P. putida KT2440 e A. A6 chlorophenolicus. In questo lavoro si illustra come preparare formulazioni per la liofilizzazione e il modo di indagare la meccanicaismi della sopravvivenza cellulare dopo la reidratazione caratterizzando la formulazione di usando calorimetria a scansione differenziale (DSC), misure di tensione superficiale, analisi a raggi X e microscopia elettronica e tali dati relativi ai tassi di sopravvivenza. I polimeri sono stati scelti per ottenere una struttura monomerica del rispettivo saccarosio polisaccaride simile ad una misura diversa. Usando questa configurazione metodo abbiamo dimostrato che polimeri possono supportare la sopravvivenza cellulare nel modo più efficace disaccaridi se certe proprietà fisiche della formulazione sono controllate 1.

Tabella 1 mostra i dati sulla composizione formulazione, eventi termici registrati dalla DSC durante il riscaldamento delle formulazioni congelati, la struttura dei campioni secco e la tensione superficiale delle soluzioni di formulazione. La T g &#39;di saccarosio sia stata determinata a -40 ° C 2, 3 e può essere difficile da rilevare per le concentrazioni di saccarosio inferiore al 20% w / w. L&#39;evento termico a -35 ° C è probabilmente correlato alla comparsa di g...

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

Liofilizzazione è spesso un modo semplice e conveniente per ottenere prodotti a secco di cellule batteriche vitali. Un problema del processo è la sopravvivenza delle cellule. Abbiamo dettaglio qui una procedura per indagare come sopravvivenza cellulare durante la liofilizzazione è influenzata dalle proprietà della formulazione utilizzata.

Formulazioni per la liofilizzazione di batteri e la loro influenza sulla sopravvivenza cellulare

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

Research

JoVE Journal

Biology

23.2K Views.  Swedish University of Agricultural Sciences. Liofilizzazione è spesso un modo semplice e conveniente per ottenere prodotti a secco di cellule batteriche vitali. Un problema del processo è la sopravvivenza delle cellule. Abbiamo dettaglio qui una procedura per indagare come sopravvivenza cellulare durante la liofilizzazione è influenzata dalle proprietà della formulazione utilizzata.

Video: 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.

Ottenere ematociti concentrati dal hawaiano Bobtail Squid Euprymna scolopes L&#39;osservazione e la loro aderenza ai batteri simbionti e non Symbiotic

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

Ricerca

Biologia

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.

Rilevazione di modificazioni post-traduzionali in nativo nucleosomi intatto da 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.

Analizzando la funzione di GTPasi Small Microiniezione di plasmidi in cellule epiteliali polarizzate

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.

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

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.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

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.

Strategie per l&#39;inseguimento Anastasis, una cellula di sopravvivenza fenomeno che Inverte 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 ...

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.

Un metodo standardizzato per l&#39;analisi delle cellule epatiche sinusoidali endoteliali e loro fenestrazioni da microscopia elettronica a scansione

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.

Misurazione del Tempo di sopravvivenza in<em&gt; Brachionus</em&gt; Rotiferi: Sincronizzazione di materni Condizioni

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.

Indagando i dannosi effetti di basso pressione sterilizzazione al Plasma sulla sopravvivenza di Bacillus subtilis spore utilizzando Live Cell microscopia

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.

Quantificazione di site-specific Protein lisina acetilazione e stechiometria Succinylation usando la spettrometria totale di acquisizione dati-indipendente

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