The work was funded by the Leukemia and Lymphoma Society of the USA (6105-08), a Cancer Council NSW grant (13-02), an NHMRC Senior Research Fellowship (LJB) (1042305) and project grant (1041614).

Le protocole décrit ici utilise la lignée cellulaire lymphoblastique leucémie aiguë NALM6 mais peut être appliquée à d&#39;autres types de cellules. 1. Les solutions et réactifs <ol><li> RPMI complet <ol><li> Ajouter 56 ml de sérum de veau foetal (FCS) et 5,5 ml de 200 mM de L-glutamine à une bouteille de milieu RPMI-1640 500 ml. </li></ol></li><li> BrdU Stock Solution <ol><li> Préparer 32,5 mM BrdU (10 mg / ml) dans une solution saline tamponnée au phosphate de Dulbecco (DPBS). </li></ol></li><li> BrdU complet RPMI <ol><li> Ajouter 6,2 ul de BrdU solution stock à 10 ml de RPMI complet. </li></ol></li><li> Solution de DNase <ol><li> Préparez 1 mg DNase / ml dans du DPBS. </li></ol></li><li> La coloration du tampon <ol><li> Préparer 3% de FCS inactivé par la chaleur et de l&#39;azoture de sodium à 0,09% dans du DPBS. </li></ol></li><li> Reportez-vous à la liste de matériaux pour les définitions de Fixation Buffer, Perméabilisation tampon et le tampon de lavage. </li></ol> 2. Cellules NonTe: Les cellules ont été cultivées pendant pas plus de 6 mois. Cette méthode est directement adaptable à toute lignée de cellules non-adhérentes avec des ajustements à la densité des cellules et des milieux de culture. Utiliser les cellules qui croissent de façon exponentielle au début de l&#39;expérience. <ol><li> Maintenir cellules NALM6 en flacons T-75 de la culture dans RPMI complet. Effectuez toutes les étapes dans des conditions stériles en utilisant une classe II Cabinet prévention des risques biotechnologiques. <ol><li> Maintenir cellules NALM6 entre 1-2 x 10 6 cellules par ml en divisant la culture trois fois par semaine. </li><li> Incuber à 37 ° C dans 5% de CO2 dans l&#39;air. </li></ol></li></ol> 3. Pulse marquage des cellules avec BrdU ATTENTION: Manipulez BrdU avec précaution car il est un mutagène potentiel et tératogène. <ol><li> Centrifuger les cellules à 150 g pendant 5 min. Remarque: Transfert de cellules dans un milieu frais améliore la reproductibilité des résultats. </li><li> Effectuer une numération cellulaire et remettre les cellules en RPMI complet à 2 x 10 6 cElls / ml. </li><li> Diluer les cellules 1 à 2 avec du milieu RPMI complet BrdU production d&#39;une concentration cellulaire finale de 1 x 10 6 cellules / ml. </li><li> Incuber à 37 ° C avec 5% de CO 2 pendant 45 minutes, puis diluer les cellules dans une 10 avec du milieu RPMI complet. Centrifuger les cellules à 150 g pendant 5 min et soigneusement jeter tout le surnageant. </li><li> Resuspendre les cellules dans un petit volume (~ 100 ul) de RPMI complet, effectuer une numération des cellules et ajuster à 1 x 10 6 cellules / ml. </li><li> Pipette 1 ml de cellules dans les puits d&#39;une plaque de 48 puits. Pipette de 1 ml de DPBS dans tous les puits inoccupés pour obtenir des résultats plus reproductibles. </li><li> Incuber à 37 ° C dans 5% de CO2 dans l&#39;air pendant points temporels souhaités, ici 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, et 24 heures. Remarque: La longueur de temps dépendra de ce que la conception expérimentale vise à mesurer. </li><li> Transférez toutes les cellules dans des tubes FACS aide d&#39;une pipette. Rincez le bien séquentiellement avec 1 ml volumes de PBS à un volume total final de 5 ml. </li><li> Centrifuger à 150 g pendant 5 min et retirez soigneusement tous les surnageant. Cellules sont prêtes pour la coloration, effectuer cette (section 4) immédiatement. </li></ol> 4. coloration des cellules Remarque: si la coloration de surface des cellules est nécessaire exécuter avant la fixation, en veillant à ce que les cellules sont maintenues à 4 ° C tout au long. <ol><li> Resuspendre les cellules dans 100 ul de tampon de coloration (pour la coloration en surface en option, ajouter le volume recommandé de l&#39;anticorps à des antigènes de surface et incuber pendant 30 min à 4 ° C). </li><li> Ajouter 1 ml de tampon de coloration, centrifuger pendant 5 min à 150 xg et jeter le surnageant. Nota: Les anticorps spécifiques, la concentration, le temps d&#39;incubation, etc. variera en fonction des objectifs expérimentaux spécifiques. </li><li> Fixation et Perméabilisation <ol><li> Resuspendre les cellules dans 100 ul de tampon de fixation et incuber pendant 15 min à température ambiante. </li><li> Ajouter 1 ml otampon de lavage de f, centrifugeuse pendant 5 min à 150 xg et jeter le surnageant. </li><li> Resuspendre les cellules dans 100 ul de tampon de perméabilisation et incuber les cellules pendant 10 min sur de la glace. </li><li> Ajouter 1 ml de tampon de lavage, centrifuger pendant 5 min à 150 xg, et jeter le surnageant. </li><li> Resuspendre les cellules dans 100 ul de tampon de fixation par tube et incuber pendant 5 minutes à température ambiante. </li><li> Ajouter 1 ml de tampon de lavage, centrifuger pendant 5 min à 150 xg, et jeter le surnageant. Remarque: Le protocole peut être mis en pause ici si nécessaire. Les cellules fixées sont stables pendant plusieurs jours à 4 ° C si on remet en suspension dans du tampon de coloration. Retirez le tampon de coloration après centrifugation avant de procéder. </li></ol></li><li> Traitement DNase <ol><li> Resuspendre les cellules dans 100 ul de solution de DNase (30 pg de DNase / 10 6 cellules) et incuber les cellules pendant 1 heure à 37 ° C. </li><li> Ajouter 1 ml de tampon de lavage, centrifuger à 150 g pendant 5 min et éliminer le surnageant. </li&gt; </ol></li><li> Anticorps Coloration Note: La coloration de marqueurs intracellulaires autres que BrdU peut être réalisée simultanément avec la coloration de BrdU. <ol><li> IMPORTANT: Préparer des témoins de compensation comprenant des cellules non colorées et les cellules marquées avec chaque fluorochrome unique. Idéalement, utilisez les mêmes anticorps pour les contrôles de rémunération que celles utilisées dans les tubes expérimentaux. Cependant, si cela est possible, des anticorps contre des antigènes de substitution hautement exprimés conjugués à la même fluorochrome. </li><li> Remettre en suspension les cellules dans 50 ul de tampon de lavage et ajouter 1 pl / 10 6 cellules d&#39;anticorps BrdU. Remarque: les anticorps directement conjugués à d&#39;autres antigènes intracellulaires spécifiques peuvent également être ajoutés. REMARQUE:. Les anticorps anti-histone H3 phosphorylée sur Ser10 peuvent être utilisées pour établir une discrimination entre les cellules dans G2 et M, l&#39;histone H3 est phosphorylée sur Ser10 lors de la mitose 10 Les anticorps anti-cdc2 phosphorylés sur Tyr15 peut être utilisé pour détecter des cellules qui HAVe engagée à la mitose. 11 </li><li> Incuber les cellules pendant 20 min à température ambiante. </li><li> Ajouter 1 ml de tampon de lavage, les cellules de centrifugation à 150 g pendant 5 min et éliminer le surnageant. </li></ol></li></ol><ol start="6"><li> Stain ADN pour l&#39;analyse du cycle cellulaire <ol><li> Desserrer pastille et ajouter 20 ul de la solution de 7-AAD (0,25 ug). Remarque: Il est essentiel d&#39;utiliser une quantité constante de 7-AAD / cellule. </li><li> Remettre en suspension les cellules dans 1 ml de tampon de coloration. </li></ol></li></ol> 5. Collecte de données de cytométrie en flux Remarque: La machine requis dépendra du nombre et de la nature des fluorochromes utilisés. <ol><li> Recueillir les paramètres suivants: FSC-A, SSC-A, FSC-H (FSC-W peuvent être utilisés à la place de FSC-H) et 7-AAD fluorescence sur une échelle linéaire. Recueillir le canal APC sur une échelle logarithmique. Recueillir tous les canaux supplémentaires nécessaires pour l&#39;évaluation de surface ou internes des étiquettes en utilisant une échelle logarithmique. </li><li> Effectuer comcompensation de chevauchement des signaux dans les spectres d&#39;émission observée entre les différents fluorochromes avant d&#39;analyser les échantillons. Note: La plupart des cytomètres de flux effectueront automatiquement. </li><li> Recueillir au moins 10 000 événements pour chaque échantillon. </li></ol> 6. Analyse de cytométrie en flux de données Remarque: FlowJo a été utilisé dans cette étude pour cytométrie de flux de données, mais d&#39;autres logiciels peuvent également être utilisés. La stratégie de déclenchement est illustré sur la figure 1. <ol><li> Identifier la population de cellules viables en utilisant les paramètres FSC-A et SSC-A. </li><li> Dans cette population exclure doublets et d&#39;agrégats à l&#39;aide de FSC-A et FSC-H (FSC-W peut également être utilisée ici). </li><li> Dans cette population définir un tracé de points en utilisant 7-AAD sur l&#39;axe des x et BrdU-APC sur l&#39;axe-y. </li></ol><img alt="Figure 1" src="/files/ftp_upload/52840/52840fig1highres.jpg" width="700" /> Figure 1: Stratégie Gating Gauche pa.nel: cellules non synchronisées sont présentés sur une FCS-A vs SSC-A dot plot. La population de cellules viables est identifié par la grille présentée. Panneau central: cellules bloquées à partir du panneau gauche sont indiqués sur une FSC-A vs FSC-H tracé de points (FSC-W peut être utilisé à la place de la hauteur). Doublets et les agrégats sont identifiés et exclus par la porte montré. Panneau de droite: cellules gated partir de la date de l&#39;exclusion de doublet dans le panneau central sont présentés sur un 7-AAD vs APC-A dot plot. L&#39;anticorps est marqué avec BrdU APC permettant l&#39;identification des cellules qui ont incorporé la BrdU au cours de l&#39;étiquetage d&#39;impulsions. 7-AAD fournit des informations sur la teneur en ADN. La porte supérieure définit cellules positives pour BrdU et donc en phase S au moment de l&#39;impulsion de BrdU, la porte en bas à gauche, les cellules en G 0/1 et la porte en bas à droite ceux de G 2 / M. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig1highres.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une plus grande version de ce chiffre.</a> <ol start="4"><li> Cell Cycle A<html> nalyse <ol><li> Ouvrez le premier fichier de données et porte sur les cellules dans la porte de l&#39;exclusion de doublet. </li><li> Analyser cette population pour la distribution du cycle cellulaire (situé sous les plates-formes de logiciels FloJo) et utiliser le modèle de Dean-Jett-Fox. </li><li> Procurez-vous les positions du G 0/1 et G 2 / M pics en utilisant créer portes. </li><li> Porte sur la BrdU des cellules positives et sous cette même population à l&#39;analyse du cycle cellulaire. </li><li> Fournir les positions pour le G 0/1 et G 2 / M pics en appliquant les mêmes portes de créer des portes et la mise contraintes (en utilisant les portails créés) pour les positions du G 0/1 et G 2 / M pics. Ceci est illustré dans les deux premiers panneaux de la figure 2. Remarque: Autre logiciel peut également être utilisé pour analyser les données et les instructions varie en conséquence. </li></ol></li></ol>840 / 52840fig2highres.jpg &quot;width =&quot; 700 &quot;/&gt; Figure 2:. Progression du cycle cellulaire Le premier panneau (toutes les cellules) est fermée sur la population de cellules défini par la porte d&#39;exclusion de doublet. Cette population a été affichée dans un histogramme avec 7-AAD sur l&#39;axe-X. Le pic du pic G 0/1 est indiqué par la flèche en dessous de l&#39;axe. Dans les panneaux ultérieures des cellules positives à la BrdU ont été fermée comme sur la figure 1. La valeur de la position de 0/1 G obtenue lorsque gating de la porte d&#39;exclusion de doublet est appliquée aux cellules BrdU positives gated logiciel au sein du cycle cellulaire FlowJo. Chaque panneau a été fermée subséquent sur ​​la population BrdU positives comme représenté sur la figure 1 et la position du pic G 0/1 sur la base de la valeur obtenue dans l&#39;analyse de l&#39;ensemble de la population comme indiqué dans les deux premiers panneaux. Utilisation de la fraction BrdU négatif pour identifier l&#39;emplacement de la population G 0/1 pour les cellules positives à la BrdU dans la même sample contrôle des différences légères dans l&#39;intensité de la tache d&#39;ADN entre les échantillons. Le numéro indiqué sur chaque panneau représente le temps écoulé depuis l&#39;impulsion BrdU terminé. Les phases du cycle cellulaire calculées sont indiquées en vert ombragé. <a href="https://www.jove.com/files/ftp_upload/52840/52840fig2highres.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a>

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The authors have nothing to disclose.

La capacité d&#39;analyse du cycle cellulaire est importante pour la compréhension de la biologie du cancer et le mécanisme d&#39;action de ces deux médicaments et les gènes qui influencent la prolifération cellulaire et la croissance. Bien qu&#39;il existe une multitude de dosages qui mesurent la prolifération cellulaire aurait, dont la majorité ne fournissent une mesure qui indique les cellules numériques présents. Ceux-ci comprennent des dosages qui mesurent le nombre de cellules par visualisation directe e...

L&#39;évaluation des caractéristiques du cycle cellulaire et les changements qui se produisent dans les cellules au cours de la progression du cycle cellulaire est fondamentale pour comprendre de nombreux aspects de la biologie, en particulier la biologie du cancer. De nombreux agents de développement pour le traitement de tumeurs malignes ont des effets profonds sur la progression du cycle cellulaire ou induire la mort cellulaire par l&#39;intermédiaire de mécanismes dépendants-du cycle cellulaire. Afin d&#39;étudier la dynamique du cycle cellulaire ou des cellules dans une phase particulière du cycle cellulaire, il est usuel de synchroniser les cellules. Cependant, les méthodes de synchronisation peuvent avoir des effets néfastes sur les cellules étudiées, potentiellement confondant les résultats obtenus. 1 analyse Récemment, l&#39;utilisation de protéines marquées par fluorescence qui ne sont présents à des phases particulières du cycle de cellules ont permis la progression du cycle cellulaire dans des cellules individuelles au fil du temps 2, cependant les cellules à étudier besoin d&#39;être génétiquement manipulée pour exprimer ces protéines marquées, ce qui limite leur utilisation à des systèmes dans lesquels il peut être luily atteint. Le cycle cellulaire est constitué de deux phases actives: la phase de synthèse (S), où l&#39;ADN est répliqué et la mitose (M) où la division cellulaire a lieu. Ces phases sont séparées par trois phases de l&#39;écart, G 0, G 1 et G 2. G 0 ou quiescence, est une phase de repos où la cellule a laissé le cycle, G 1 est où les cellules augmentent de taille avant replication de l&#39;ADN et G 2 dans laquelle la croissance cellulaire continue entre l&#39;achèvement de la replication de l&#39;ADN, mais avant la division cellulaire. La progression à travers le cycle cellulaire est régulée par un certain nombre de points de contrôle. Le G 1 point de contrôle est activé lorsque les conditions environnementales ne sont pas favorables à la synthèse d&#39;ADN et empêche l&#39;entrée en phase S. La phase checkpoint ou de retard intra-S peuvent être déclenchées par des dommages de l&#39;ADN qui peuvent entraîner des fourches de réplication bloquées. Pendant G 2 de la fidélité de l&#39;ADN répliqué est confirmée et si le dommage est détecté, le G 2 </sub&gt; checkpoint est activée en permettant réparation de l&#39;ADN avant la division cellulaire. Un point de contrôle finale lors de la mitose assure que chromatides ont été correctement alignés à la plaque mitotique sorte que la division cellulaire peut être complété avec succès. 3 L&#39;activation de ces points de contrôle est communément utilisé pour synchroniser des populations cellulaires. points de contrôle du cycle cellulaire peuvent être activés par un certain nombre de facteurs, mais dans la biologie du cancer le plus commun est la détection des lésions de l&#39;ADN. La réponse aux dommages de l&#39;ADN est initiée par la PI3-kinase comme le télangiectasie kinases de l&#39;ataxie et Rad3 liées (ATR) et protéine atm (ATM) qui activent les kinases effectrices aval Chk1 et Chk2, respectivement. 3 Une gamme d&#39;événements active Chk1 y compris l&#39;impasse fourches de réplication, réticulations d&#39;ADN, et ultraviolet dégâts d&#39;irradiation tout Chk2 est principalement activés par cassures double-brin. La méthode habituelle pour étudier l&#39;effet de conditions modifiées de la durée du cycle cellulaire is pour synchroniser les cellules dans une phase particulière du cycle cellulaire. 1 Ceci peut être réalisé par plusieurs procédés. Les cellules peuvent être physiquement séparés en fonction de la taille, la densité, la diffusion latérale (granularité), et des marqueurs d&#39;expression de surface cellulaire. Plus concrètement, les cellules peuvent être synchronisées par des moyens chimiques. Plusieurs agents tels que la thymidine, l&#39;hydroxyurée et la cytosine arabinoside peuvent être utilisés pour inhiber la synthèse d&#39;ADN dans la phase S du cycle cellulaire résulte en une accumulation de cellules en phase S qui continuent après cyclage les agents sont enlevés. Les cellules traitées avec le nocodazole, ce qui empêche la formation du fuseau mitotique, avec un arrêt G 2 - phase M ou la teneur en ADN. Élimination de sérum à partir des résultats de milieu de culture dans l&#39;accumulation de cellules en G 0 phase. Le rajout des éléments nutritifs dans le sérum de culture relance le vélo normal des cellules. Cependant, toutes ces méthodes de synchronisation normale interférer avec le vélo et la croissance des cellules et peut Result dans la mort cellulaire significative. Synchronisation des cellules de leucémie lymphoblastique aiguë est particulièrement difficile et ces cellules ne se prêtent pas à la manipulation génétique. Le procédé décrit ici permet l&#39;évaluation de la dynamique du cycle cellulaire et l&#39;étude des cellules en phases particulières du cycle cellulaire sans synchronisation traditionnel ou une modification génétique. Cette méthode peut également être utile pour d&#39;autres types lorsque la modification génétique et les procédures de synchronisation traditionnels ne sont pas facilement obtenus cellulaires. La méthode est basée sur l&#39;utilisation de longue date de bromodésoxyuridine (BrdU) l&#39;incorporation, qui a très peu d&#39;impact sur ​​la croissance à court terme et la prolifération des cellules. 4 protocoles de BrdU établis profitent de l&#39;incorporation de BrdU dans l&#39;ADN nouvellement synthétisé pendant la phase S . Ceci marque en permanence des cellules comme ayant été en phase S pendant l&#39;exposition au BrdU. Cette population peut être identifié à des temps plus tard par coloration pour BrdU Incorporation et ainsi agir comme une population synchronisé qui peut être suivie et évaluée dans le temps permettant l&#39;étude des effets des médicaments sur le transit du cycle cellulaire. BrdU besoin d&#39;être exposée avant la coloration des anticorps, généralement obtenus après traitement à l&#39;acide ou DNase. 6,7 En utilisant la cytométrie en flux pour détecter la BrdU incorporée permet l&#39;inclusion de marqueurs supplémentaires. Le plus important est l&#39;utilisation de colorants pour mesurer la teneur en ADN, ce qui permet l&#39;évaluation de la distribution de phase du cycle cellulaire des cellules qui étaient en phase S au début de l&#39;étude. 8 surface ou intracellulaires des antigènes outre supplémentaires peuvent également être étudiés. 9 Ces peuvent se rapporter à des événements du cycle cellulaire telles que Ki67 ou fonctions cellulaires à apparemment indépendants tels que des marqueurs de l&#39;apoptose tels que la caspase-3 clivée. Les applications potentielles sont limitées par l&#39;imagination de l&#39;enquêteur.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>APC BrdU Flow Kit</td>
			<td>BD Biosciences</td>
			<td>552598</td>
			<td>Contains BrdU antibody, 7-AAD and BD Cytofix/Cytoperm 
			Buffer (referred to as Fixation Buffer)</td>
		</tr>
		<tr>
			<td>BD Cytoperm Permeabilization Buffer Plus</td>
			<td>BD Biosciences</td>
			<td>561651</td>
			<td>Referred to as Permeabilization buffer</td>
		</tr>
		<tr>
			<td>BD Perm/Wash Buffer</td>
			<td>BD Biosciences</td>
			<td>554723</td>
			<td>Referred to as Wash buffer</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma</td>
			<td>D-4513</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon 12 x 75-mm FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>554656</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR FORTESSA flow cytometer</td>
			<td>BD Biosciences</td>
			<td>FORTESSA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman</td>
			<td>Gilson</td>
			<td>P2, P20, P100, P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o L-Gln 500 ml</td>
			<td>Lonza</td>
			<td>12-167F</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>FisherBiotec</td>
			<td>FBS-7100113</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-2&#8242;-deoxyuridine</td>
			<td>Sigma</td>
			<td>B5002-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon TC 150cm2 vented Flasks</td>
			<td>BD Biosciences</td>
			<td>355001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 25mL</td>
			<td>Greiner</td>
			<td>760180</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 200&#181;L</td>
			<td>Interpath</td>
			<td>24700</td>
			<td></td>
		</tr>
		<tr>
			<td>Aersol Pipettes 1mL</td>
			<td>Interpath</td>
			<td>24800</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Spintron</td>
			<td>GT-175R</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Binder</td>
			<td>C 150</td>
			<td></td>
		</tr>
		<tr>
			<td>AF488 anti-Histone H3 Phospho (Ser10) Antibody</td>
			<td>Cell Signalling</td>
			<td>9708S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb</td>
			<td>Cell Signalling</td>
			<td>2197S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Chk1 (Ser345) (133D3) Rabbit mAb</td>
			<td>Cell Signalling</td>
			<td><a name="RANGE!C23">2348S</a></td>
			<td></td>
		</tr>
		<tr>
			<td>NALM6&#160;</td>
			<td>DSMZ</td>
			<td>ACC-128</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Cette méthodologie peut être utilisée pour obtenir une série d&#39;informations. Quelques applications sont décrites ici. L&#39;évaluation de la durée du cycle cellulaire Pour déterminer le temps nécessaire pour les cellules de transit à travers le cycle cellulaire, les cellules sont récoltées à divers points de temps suivants l&#39;impulsion de BrdU. Les intervalles entre les évaluations peuvent être adaptés à des cellules particulières en cour...

Watch this Scientific Journal Video about Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization at JoVE.com

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

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

Ce protocole décrit l&#39;utilisation de bromodésoxyuridine (BrdU) absorption pour permettre le suivi temporel des cellules qui étaient en phase S à un moment précis dans le temps. Addition de colorants d&#39;ADN et de l&#39;étiquetage d&#39;anticorps facilite l&#39;analyse détaillée du sort des cellules en phase S au temps plus tard.

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 ...

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19.0K vues.  Westmead Millennium Institute for Medical Research and University of Sydney.  Ce protocole décrit l'utilisation de bromodésoxyuridine (BrdU) absorption pour permettre le suivi temporel des cellules qui étaient en phase S à un moment précis dans le temps. Addition de colorants d'ADN et de l'étiquetage d'anticorps facilite l'analyse détaillée du sort des cellules en phase S au temps plus tard. 

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

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purification cytométrie en flux des cellules de souris méiotique

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

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Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Mesure cinétique cellulaire avec la progression du cycle marquage métabolique et cytométrie de flux

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimisé méthodes de modélisation de coloration et de prolifération pour la surveillance division cellulaire en utilisant des colorants Suivi cellulaires

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Suivi cellule à l&#39;aide protéines photoconvertible cours de développement poisson zèbre

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Techniques pour l&#39;analyse des vésicules extracellulaire par cytométrie en flux

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

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.

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 ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED Thermo Flow - Optogenetics Combinant avec cytométrie de flux

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Détermination de la surface de la cellule relative et d&#39;expression totale de Recombinant Canaux ioniques par cytométrie en flux

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analyse des suspensions de cellules isolées de tissus solides par Spectral cytométrie en flux

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

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 ...