The authors thank members of the Shapiro lab and Erin Schrader for comments on the manuscript. The authors acknowledge financial support from: NIH postdoctoral fellowship F32 GM100732 to JMS and NIH grants R01 GM51426 and R01 GM32506 to LS.

1. Synchrony à grande échelle - Optimal pour Western Blot, puces à ADN / ARN-Seq, et d&#39;autres essais intensifs Matériel <ol><li> De un congélateur actions ou une plaque, cultiver un 5 ml O / N de la culture de la souche NA1000 en secouant à 28 ° C dans un milieu PYE. </li><li> Inoculer 0,5 ml des cellules de l&#39;étape 1 dans 25 ml de M2G (tableaux 1-2) et agiter à 28 ° C jusqu&#39;à ce que la culture atteint une DO 600 entre 0,5 et 0,6. </li><li> Ensemencer les cellules dans 1 L de M2G et agiter à 28 ° C. </li><li> Une fois DO600 atteigne 0,5 à 0,6, de confirmer la présence de cellules en utilisant swarmer liquide monté microscopie de phase. Spot 1 pi de cellules sur une lame de verre, couvrir avec une lamelle, et l&#39;image par microscopie de phase. Confirmer la présence de cellules Grouillant en visualisant les cellules de natation rapidement dans la population. </li><li> Spin cellules pendant 15 min à 7 KXG à 4 ° C dans un rotor JA-10. </li><li> Jeter le surnageant et ajouter 180 ml de M2 ​​froide (tableaux 1-2) et res doucementuspend toutes les cellules en utilisant une pipette sérologique. Jeter cellules faiblement granulés; ce sont des cellules predivisional et harcelées. </li><li> Ajouter 60 ml de solution de silice colloïdale froide (ne oubliez pas de mélanger la suspension de silice colloïdale avant d&#39;ajouter aux cellules) et bien mélanger la suspension cellulaire. </li><li> Verser la suspension de cellules dans 30 ml de huit tubes et centrifuger pendant 30 min à 6,4 KXG à 4 ° C dans un rotor JA-20. NOTE: Il faut voir deux bandes distinctes; la bande de swarmer est la bande inférieure tandis que les cellules traquées / predivisional sont dans la fourchette supérieure (figure 1B). </li><li> Aspirer soigneusement la bande supérieure et enlever le liquide à ~ 1 cm au-dessus de la bande de swarmer (la bande inférieure). </li><li> (Critique) aide d&#39;une pipette Pasteur, retirez soigneusement la bande de swarmer et la placer dans un tube propre. Pour laver la silice colloïdale, en haut du tube hors M2 avec froid et essorage pendant 10 min à 6,4 KXG à 4 ° C dans un rotor JA-20. </li><li> Eliminer délicatement le surnageant et remettre en suspension leles cellules dans 20 ml de M2 ​​froid et centrifugation pendant 10 min à 6,4 KXG à 4 ° C dans un rotor JA-20. </li><li> La suspension de toutes les pastilles dans 30 ml de M2 froid et mesurer l&#39;OD 600 en utilisant un spectrophotomètre et vierge à l&#39;aide du milieu M2 froid. Enregistrer 1 pl pour l&#39;imagerie de phase pour vérifier swarmer cellules; 90-95% des cellules doit être swarmers. </li><li> Centrifuger les cellules pendant 5 min à 6,4 KXG à 4 ° C dans un rotor JA-20. Resuspendre les cellules dans 28 ° C M2G moyenne de sorte que le A 600 est ~ 0,3 à 0,4 et commencer agitation à 28 ° C. NOTE: Les rendements typiques sont entre 30 et 60 ml de swarmer culture cellulaire à partir de cellules non synchronisées 1L. </li><li> Commencez à prendre des points de temps (la culture de type sauvage prendra environ 135-140 minutes de diviser) 8,9. A chaque point de temps, de mesurer la DO 600. Vérifiez que la DO 600 après la division est d&#39;environ 2X la DO initiale 600. </li><li> Pour blot ou expression génique essais occidentaux, retirez aliquotes de 1 ml de la culture aux points de temps désirés, ralentit à la vitesse max dans une centrifugeuse de table pendant 30 sec, rapidement décanter ou aspirer le milieu, et le flash geler le culot cellulaire dans l&#39;azote liquide. Stocker les cellules à -80 ° C jusqu&#39;à l&#39;analyse en aval. </li></ol> 2. Synchrony à petite échelle - Optimal pour Microscopie <ol><li> De un congélateur actions ou une plaque, grandir à 5 ml O / N secouant la culture à 28 ° C dans M2G. </li><li> Diluer dans 15 ml de M2G (tableaux 1-2) et de croître jusqu&#39;à la mi-log (DO600 = 0,5-0,6). </li><li> Spin à 6,4 KXG pendant 10 min à 4 ° C dans un rotor JA-20, et de remettre en suspension dans 1 ml froides M2 (tableaux 1-2) et transférer dans un tube à centrifuger de 2 ml. </li><li> Centrifuger à 15 KXG pendant 3 min dans un tube de microcentrifugeuse pour sédimenter les cellules, aspirer le surnageant, le culot a mis sur de la glace, et remettre en suspension dans 900 ul de M2 ​​froid. </li><li> Ajouter 900 pi de PVP froid revêtu silice colloïdale et de spin pendant 20 min à 15 KXG à 4 ° C dans un microTube rocentrifuge. </li><li> (Critique) Aspirer ou une pipette du haut traqué / predivisional bande de cellules et de recueillir le fond swarmer bande dans un nouveau tube. </li><li> Laver les cellules deux fois swarmer dans 1 ml de M2 ​​froid tout en centrifugeant à 15 KXG pendant 3 min. </li><li> Avant l&#39;essorage final, déplacer les cellules dans un 1 ml tube à essai en verre pré-réfrigérés et mesurer la DO 600 de cellules par rapport à une ébauche de M2. </li><li> Reprendre le culot cellulaire final dans 28 ° C M2G à une DO entre 0,3 à 0,4 et secouez cellules / roll à 28 ° C. REMARQUE: Les rendements typiques sont comprises entre 2 et 4 ml de culture de cellules swarmer. </li><li> Pour les expériences de microscopie, au souhaitée points temporels lieu 1 pi de cellules sur un tampon agarose M2G pour l&#39;imagerie. </li></ol>

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The authors have nothing to disclose.

The bacterial cell cycle is a fundamental process in life and is important for the study of growth and as a target for next generation antibiotics. Here, we detailed the rapid synchronization procedures for C. crescentus NA1000, a model organism for the study of the bacterial cell cycle and asymmetric cell division. This method is amendable to western blot, gene expression profiling, and fluorescence microscopy assays to investigate the spatiotemporal regulation of the bacterial cell cycle. 
The protocol is quick and yields healthy synchronized cells. Other synchronization methods require long starvation of cells and rely on the stringent response to arrest the cell cycle in the same state, leading to cells that are dissimilar to cultures in logarithmic growth 13. While &#8220;baby machines&#8221; and other surface attachment methods have been used successfully14,15, they typically have low yields of synchronized cells. The density centrifugation protocols presented here allow higher purity and yields of synchronized C. crescentus NA1000 swarmer cells.
Additional complications can lead to potential problems throughout the synchrony procedure. Have a liquid mount phase microscope handy to optically check the purity of cells before synchronization and to check the purity of swarmers. A good synchrony will yield >95% swarmer cells. Check the OD600 on a spectrophotometer to ensure the cells are doubling during the time course of the synchrony. 
Cells don&#8217;t synchronize as well in the presence of antibiotics or some mutations. If the strain does not carry a replicating plasmid, try to remove the antibiotics from the growth medium to improve swarmer cell yield. Cells grown in the presence of antibiotics or mutant strains will also have a slower cell cycle, so it is important to check the division time by microscopy for comparison to healthy strains. 
Avoid vortexing the cells, as this will shear off the flagella and lower the quality of the synchrony. In preparation for the synchrony protocol don&#8217;t grow the cells above OD600 0.5. This ensures healthy growth that isn&#8217;t altered by high cell density. Be careful when aspirating/pipetting off supernatants containing the stalked and predivisional cell populations. In particular, do not to touch the swarmer band which may lead to contamination and/or a loss in yield of the swarmer cells. The cell pellet will be very loose in the first wash of cells after removing them from the Colloidal silica. Be careful when removing the supernatant not to disrupt the pellet.
Occasionally NA1000 cells can lose synchronization capacity due to a loss of DNA in the prophage region of the genome 7. In this case, no swarmer band is observed or the swarmer band intensity is dramatically reduced. If possible, collect cells if present in the swarmer band and streak onto a PYE plate to re-isolate the synchronizable cells. If working with a mutant strain, it is often useful to reintroduce the mutation into a fresh batch of NA1000. Conversely, mutation of critical cell cycle regulatory proteins can, in some cases, disrupt the ability of cells to synchronize16.
M2G medium was initially described with a higher 20 mM phosphate concentration 8,17; however, current studies use a lower 10 mM concentration as presented in Table 1 18,19. Alternative to the standard M2G medium, it is possible to use richer PYE medium. Here the unsynchronized growth and growth after synchronization can be substituted for PYE.
C. crescentus NA1000 provides a valuable experimental tool to study the bacterial cell cycle. Additionally, due to the single copy of the chromosome in the swarmer cell, this bacterium has also become a powerful model to study the structure of the bacterial chromosome.

Le cycle cellulaire bactérienne contrôle à la fois la replication du génome et la division des cellules filles. Surtout, comme la résistance aux antibiotiques est une menace croissante pour la santé publique, le cycle cellulaire bactérienne présente une cible inexploitée pour le développement antibiotique. Dans la bactérie Caulobacter crescentus, chaque cycle cellulaire conduit à une division asymétrique, pour donner deux cellules filles des différentes sorts (figure 1A) 1,2. Une cellule fille hérite un flagelle et mobiles tandis que l&#39;autre fille hérite d&#39;une tige et est sessiles. Circuit intégré génétique contrôle la progression du cycle cellulaire et sort de la cellule par régulation de la transcription, phospho-signalisation, et la protéolyse régulée 3. En outre, la réplication chromosomique des cellules filles et de rendement de la séparation simultanées qui contiennent exactement une copie du chromosome 4. Fait important, ces deux types de cellules peuvent être séparés rapidement par colloL&#39;Idéal particule de silice densité centrifugation dans la souche synchronisable NA1000 7.5 permettant l&#39;isolement des cellules swarmer du reste de la population avec des rendements élevés (figure 1B). Isolé swarmer cellules alors procéder de manière synchrone par division cellulaire asymétrique. Ici, nous détaillons le protocole utilisé pour synchroniser Caulobacter souche NA1000. Nous fournissons des protocoles et des conseils de dépannage communes pour deux petite et à grande échelle des synchronisations. Cette procédure expérimentale fournit un outil puissant pour interroger le contrôle spatio-temporelle du cycle cellulaire et Caulobacter destin cellulaire.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PVP Coated Colloidal Silica (Percoll)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4937</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colloidal Silica (Ludox AS-40)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">420840</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">JA10 Rotor</td>
			<td style="width:71px;">Beckman-Coulter</td>
			<td style="width:119px;">369687</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">JA20 Rotor</td>
			<td style="width:71px;">Beckman-Coulter</td>
			<td style="width:119px;">334831</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ferrous Sulfate Chelate Solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>F0518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30 mL Centrifuge Tubes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">8445</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Na2HPO4</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0720-1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KH2PO4</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH9268-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4Cl</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">0621-500g</td>
			<td></td>
		</tr>
	</tbody>
</table>

synchronization of caulobacter crescentus for investigation of the bacterial cell cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronisation donne typiquement deux bandes de cellules (figure 1b): la bande de swarmer, qui a une densité plus élevée, et un / predivisional bande cellulaire pétiole de densité inférieure. Pour assurer des contrôles communs de synchronisation efficace comprennent la surveillance de l&#39;OD 600 et de mesurer les niveaux de protéines CTRA par western blot à des moments du cycle cellulaire distincts. La DO 600 devrait augmenter d&#39;environ deux fois au cours du cycle cellulaire (Figure 2). Le western blot pour le maître régulateur du cycle cellulaire CTRA est un contrôle utile pour vérifier une bonne synchronisation (Figure 2). Ctra est utilisé dans la cellule de swarmer pour bloquer la réplication d&#39;ADN et se dégrade lors de l&#39;apparition de la 10 replication de l&#39;ADN. CTRA est ensuite synthétisé plus tard dans le cycle cellulaire et active la transcription d&#39;une multitude de gènes du développement, y compris de nombreux composants de la flagelle 11,12. Un synchronie retenu aura tson mode d&#39;oscillation du taux de protéines CTRA. <img alt="Figure 1" src="/files/ftp_upload/52633/52633fig1.jpg" /> Figure 1. Le cycle cellulaire de Caulobacter crescentus. (A) Une schématique de bande du cycle cellulaire swarmer. Les cellules se différencient en cellules Grouillant traquées compétentes de réplication en rétractant pili, éjecter flagelles, et initier la réplication de l&#39;ADN. Les cercles et thêta structures représentées à l&#39;intérieur des lignes de cellules représentent chromosomes de repos et de réplication. Cellules puis progressent dans le cycle cellulaire construire un seul flagelle à l&#39;opposé de la tige. En cas de division, deux types de cellules uniques sont générés, une réplication bloqué mobiles swarmer cellulaire et une réplication cellulaire compétente traqué stationnaire. (B) Les résultats représentatifs pour la centrifugation de densité. Faible densité de harcèlement et de predivisional cellules flottent près du haut de la pente tandis dense swarmer cellules finissent vers le fond du tube. Barres d&#39;échelle sont une um dans les images de microscopie de phase. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52633/52633fig1large.jpg" target="_blank">Se il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 2" src="/files/ftp_upload/52633/52633fig2.jpg" /> Figure 2. Les résultats représentatifs pour une synchronisation réussie swarmer cellulaire. La masse cellulaire mesurée par OD 600 devrait augmenter lentement et, finalement, deux fois tout au long de l&#39;essai. Aliquotes de 1 ml ont été introduits à la pipette dans des cuvettes en matière plastique à partir d&#39;un grand synchronie de la cellule et la DO 600 mesurée en utilisant un spectrophotomètre à des points de temps indiqués. En outre, le cycle cellulaire maître protéine régulatrice Ctra doit être présent dans la cellule swarmer pour bloquer la réplication d&#39;ADN, suivie d&#39;une dégradation rapidecoïncide avec l&#39;initiation de la réplication de l&#39;ADN. Ctra est ensuite régénéré plus tard dans le cycle de cellule pour activer l&#39;expression de nombreux gènes, y compris le développement et flagellaires pili composants critiques. Transferts de Western pour la maîtrise du cycle cellulaire protéine régulatrice CTRA ont été réalisées en prenant aliquots de 1 ml d&#39;une synchronie à grande échelle. Les cellules ont été remises en suspension dans 250 ul de tampon d&#39;échantillon par la DO 600 Laemmlli, séparé sur un TRIS-GLY PAGE 10%, transférés sur du PVDF, et transférés avec un anti-anticorps Ctra. Procédures de synchronie échoué conduisent à CTRA transferts de western sans changement dans les niveaux de protéines. α-Ctra anticorps a été incubé à 12 une dilution de 1: 10 000 pendant 1,5 h à 3% de lait TBST et on lave 3 fois dans du TBST. Goat-α-lapin secondaire a ensuite été ajoutée au 1: 10 000 dilution dans 3% TBST de lait pendant 1 heure, lavé trois fois avec du TBST et imagé sur film en utilisant un kit de détection chimioluminescent. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52633/52633fig2large.jpg" target="_blank">Se il vous plaît</a>cliquez ici pour voir une version plus grande de cette figure. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Na 2 HPO 4 </td><td> 17,4 g </td></tr><tr><td> KH 2 PO 4 </td><td> 10,6 g </td></tr><tr><td> NH 4 Cl </td><td> 10 g </td></tr><tr><td> H 2 O </td><td> Remettre en suspension dans 1 L &amp; autoclave </td></tr></tbody></table> Tableau 1. 20X M2 sels recette. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 20X M2 Sels </td><td> 50 ml </td><td></td></tr><tr><td> 0,5 M MgSÛ4 </td><td> 1 ml </td><td></td></tr><tr><td> 20% de glucose </td><td> 10 ml </td><td> Substitut pour H 2 O dans M2 </td></tr><tr><td> Sulfate ferreux chélate Solution </td><td> 1 ml </td><td></td></tr><tr><td> 0,1 M de CaCl2 </td><td> 5 ml </td><td> Ajouter dernière AVprécipitations oid </td></tr><tr><td> H 2 O </td><td> Remplir jusqu&#39;à 1 L </td><td></td></tr><tr><td> Filtre stérile avec filtre de 0,22 uM </td><td></td><td></td></tr></tbody></table> Tableau 2. M2 et M2G recette.

Watch this Scientific Journal Video about Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle at JoVE.com

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Synchronisation de<em&gt; Caulobacter crescentus</em&gt; Des enquêtes sur le cycle cellulaire bactérienne

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

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Nanyang Technological University

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Biology

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

11.8K vues.  Stanford University School of Medicine. Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Vidéo: Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Electroporation de mycobactéries

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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Biologie

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

Activités de surveillance kinase et la phosphatase dans le cycle cellulaire par Ratiométrique FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse de la situation du cycle cellulaire dans les cellules de mammifères

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

La Ex vivo Préparation isolé microvaisseaux squelettique pour les enquêtes de la réactivité vasculaire

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

De base<em&gt; Caenorhabditis elegans</em&gt; Méthodes: Synchronisation et d&#39;observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

Suivi temporel de la progression du cycle cellulaire par cytométrie en flux, sans la nécessité pour la synchronisation

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Mesures de contractilité des muscles papillaires isolés sur pour l&#39;Investigation des inotropie cardiaque chez la souris

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Une expression efficace de cellules de mammifères et en une seule étape de purification extracellulaire glycoprotéines à la cristallisation

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

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 ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Combinant la synchronisation cellulaire mitotique et microscopie confocale haute résolution pour étudier le rôle des protéines multifonctionnelles Cycle cellulaire au cours de la mitose

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

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