Gli autori sono supportati dal Consiglio di ricerca svedese, la Fondazione svedese per la ricerca strategica, la società svedese cancro, la società svedese cancro infantile, Åke Wibergs fondazione e Jeanssons fondazione.

1. Introducendo la sonda per le cellule <ol><li> Co-trasfezione delle cellule con una sonda FRET-based e una resistenza plasmide conferimento. Scegliere un metodo di trasfezione che sia efficiente in cella tipo di interesse. Per le celle U2OS, standard di fosfato di calcio metodi di trasfezione dare risultati adeguati 15. </li><li> Selezionare le celle nel antibiotico appropriato per almeno sette giorni. Ciò arricchisce la quantità di cellule che esprimono la sonda e limita la quantità di cellule con livelli di espressione tossici, o livelli di espressione che influenzano pesantemente il ciclo cellulare. </li><li> (Facoltativo) Seleziona per cloni stabili. </li><li> Usando cellule fisse o dal vivo, verificare che sia CFP e YFP sono presenti in tutte le cellule a circa lo stesso rapporto. </li><li> Nel caso in cui alcune cellule contengono solo CFP o YFP, ricombinazione è probabilmente avvenuto. Ricombinazione può avvenire sia durante la crescita batterica o dopo la transfezione della sonda e può in quest&#39;ultimo caso, dipendono dalla qualità plasmide. Ripetere p plasmideriparazione e linearizzare il plasmide prima trasfezione se il problema persiste. </li></ol> 2. Raziometrico monitoraggio FRET <ol><li> Cellule seme su piatti fondo di vetro, o scivola copertura per il montaggio in una camera. Assicurarsi che i piatti / coprioggetto sono 1,5 (170 micron di spessore). </li><li> Montare le cellule nel terreno di coltura senza rosso fenolo su un microscopio a epifluorescenza motorizzato con controllo della temperatura impostata a 37 ° C. Garantire una regolamentazione del pH dei media, sia per CO 2 oppure utilizzando CO 2-media indipendenti come Liebowitz-15. </li><li> Focus su cellule a luce trasmessa. Non guardare sulle cellule usando la luce fluorescente. </li><li> Inizio con l&#39;acquisizione di un 12 o 16 bit immagine con eccitazione YFP (YFPex) e YFP emissione (YFPem) filtri e uno specchio dicroico adatto. Usa binning massima disponibile che permette ancora la risoluzione richiesta subcellulare. Utilizzando un binning alto per diminuire il tempo di esposizione o l&#39;intensità è fondamentale per evitare di fototossicità. </li><li> MeAsure o stimare l&#39;intensità del rumore di fondo e approssimativo, misurando o stimando la media massima, minima e massima dei valori dei pixel in una zona priva di cellule. Tempo di esposizione e cambiare i filtri a densità neutra in modo che il segnale di media nella cella è di circa l&#39;intensità di fondo più cinque a dieci volte la differenza tra max e min intensità di fondo. </li><li> Verificare che le immagini non sono vicino alla saturazione. L&#39;intensità massima l&#39;immagine deve essere inferiore alla metà della gamma dinamica (per esempio max 2000 in un&#39;immagine a 12 bit) per tener conto delle differenze di intensità quando le cellule entrano mitosi. </li><li> Ripetere il passo 2,4-2,6 con eccitazione CFP e YFP filtri emissione e uno specchio dicroico adatto. </li><li> Selezionare più regioni con le cellule transfettate, escludendo le regioni utilizzate per punto 2.4 del 2.7, e garantire che l&#39;intensità massima è meno della metà della gamma dinamica in tutte le cellule. </li><li> Avviare un time-lapse esperimento acquisizione di due immagini ogni 45 min con YFPex - YFPem e CFPex - YFPem set di filtri. </li></ol> 3. Verifica e ottimizzazione delle condizioni <ol><li> Aprire le immagini acquisite e monitorare il tempo di ciclo cellulare da mitosi a mitosi per le cellule che esprimono la sonda transfettate. </li><li> Confrontare il tempo misurato ciclo cellulare ai dati pubblicati per il tipo di cellulare utilizzato. In alternativa, film untransfected cellule con l&#39;acquisizione di una immagine di luce trasmessa solo (ad esempio DIC o contrasto di fase) ogni ora per ottenere riferimento i tempi di ciclo cellulare. </li><li> Nel caso in cui il tempo da mitosi a mitosi corrisponde al riferimento tempi ciclo cellulare, passare al punto 4. In alternativa, ri-acquisire le immagini con più esposizione, più tempo o più punti di z-livelli e ripetere il punto 3. </li><li> Nel caso in cui il tempo da mitosi a mitosi non corrisponde a tempi di riferimento del ciclo cellulare, le cellule trasfettate pellicola con l&#39;acquisizione di una immagine di luce trasmessa solo (ad esempio DIC o contrasto di fase) ogni ora seguita da una sola immagine alla finedell&#39;esperimento per identificare le cellule transfettate. </li><li> Se le cellule trasfettate mostrano ancora i tempi di ciclo più lungo cellulare, ripetere il passo 1, ma trasfezione meno sonda o per selezionare i cloni stabili che contengono quantità molto basse di sonda. </li><li> Se le cellule trasfettate mostrano normali tempi di ciclo cellulare, in assenza di luce fluorescente, ripetere il punto 2, ma una minore esposizione modificando binning, i filtri a densità neutra, tempo di esposizione, e il tempo tra le immagini. </li></ol> 4. Analizzando FRET <ol><li> L&#39;analisi può essere eseguita dalla maggior parte dei software dedicati alla microscopia. Qui si descrivono come eseguire l&#39;analisi utilizzando il freeware ImageJ ( <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a> ) utilizzando il plugin Rapporto Plus ( <a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plus . html</a> ). </li><li> Aprire le immagini in ImageJ. Nel caso in cui le immagini sono nel formato di un Stac multicolorek (ad esempio Deltavision), separare i singoli canali di prima li converte in un HyperStack: Image-HyperStack-stack HyperStack e successivamente dividerli a pile singolo canale usando: Immagine-HyperStack-ridurre la dimensionalità </li><li> Moltiplicare il YFPex - pila YFPem con 3 usando: Process-Math-Moltiplicare Questo passaggio è facoltativo, in quanto è solo destinato ad aumentare la chiarezza visiva delle immagini, assicurando che i rapporti non sono nel range compreso tra 0 e 1. </li><li> Nel YFPex - pila YFPem, disegnare una regione di interesse (ROI) in una zona che è privo di cellule, ma che si trova vicino a una cella da misurare. Cellule diverse nella stessa immagine può richiedere diverse regioni, sia come sfondo e l&#39;intensità del segnale è di solito più forte al centro di un&#39;immagine. </li><li> Aggiungere il ROI al ROI manager: Analisi-tools-ROI manager. </li><li> Misurare l&#39;intensità media del ROI in entrambe le YFPex - YFPem e CFPex - pila YFPem: Analisi-Misura </em&gt;. </li><li> Ripetere in vari intervalli di tempo e verificare che intensità sfondo vicino alla cella di interesse sono simili in tutto il film. </li><li> Impostare le misure per includere l&#39;intensità minima: Analisi-Set misurazioni-Min e Max valore di grigio. </li><li> Disegnare una regione di interesse che coprono la maggior parte di una cella e misurare l&#39;intensità minima in entrambe le YFPex - YFPem e CFPex - pila YFPem. Prendere la differenza tra l&#39;intensità misurata minimale e l&#39;intensità di fondo da 4,6 e dividerlo per due. Questo fornisce una stima di partenza per un valore di ritaglio. </li><li> Rapporto + aperto. Selezionare la YFPex - YFPem e CFPex - pila YFPem. Per FRET rapporto, selezionare CFPex - YFPem come stack 1, per il rapporto invertito FRET visualizzazione sonde FRET in cui riduce l&#39;efficienza sul fosforilazione, selezionare YFPex - YFPem come stack 1. </li><li> Inserire l&#39;intensità di fondo misurata ed i valori di ritaglio. </li><li> Impostare la scala dello stack rapporto risultante e applicare un u adatto sguardop table (LUT) di visualizzare le modifiche rapporto. </li></ol> 5. Rappresentante Risultati Plk1 attività è il primo visibile nel nucleo in fase G2 e picchi durante la mitosi. La figura 3 mostra un esperimento utilizzando le impostazioni di fototossicità minime come descritto nella sezione 2. Si prega di notare che questo è un risultato rappresentativo iniziale e che le condizioni di esposizione o di tempo tra le immagini possono essere modificati per aumentare rapporto segnale rumore o di risoluzione temporale. La figura mostra in 3D che la maggioranza delle cellule che esprimono la sonda proliferare con un tempo di ciclo cellulare tra 20 e 25 ore, indicando che le condizioni di imaging e livelli di espressione della sonda non influiscono tempi del ciclo cellulare. Anche se c&#39;è rumore considerevole, il trend di Plk1 crescente attività in G2 e un picco nel mitosi 14 è chiaramente visibile (fig. 3A). L&#39;elaborazione dei dati grezzi, qui da dire filtraggio presentato come un chimografo (fig. 3B), o la quantificazione della media ra invertita<html> tio (fig. 3C) può migliorare la chiarezza. <img alt="Figura 1" src="/files/ftp_upload/3410/3410fig1.jpg" /> Figura 1. Principio di una sonda FRET a base di monitorare le attività chinasi e fosfatasi. Due fluorofori, tipicamente CFP (blu) e YFP (verde), sono collegati da una fosfo-dominio di legame (arancione) e una sequenza phosphorylatable (giallo). Fosforilazione (rosso) media il legame con i fosfo-dominio di legame, in modo da indurre un cambiamento conformazionale nella sonda. I risultati cambiamento conformazionale in una differenza nella distanza o l&#39;orientamento tra i due fluorofori, che colpisce l&#39;efficienza FRET tra CFP e YFP. FRET può essere visualizzato CFP emozionante e monitoraggio delle emissioni YFP (linee tratteggiate). <img alt="Figura 2" src="/files/ftp_upload/3410/3410fig2.jpg" /> Figura 2. Delineare schematica della procedura sperimentale. p_upload/3410/3410fig3.jpg &quot;/&gt; Figura 3. Plk1 attività è il primo visibile nel nucleo in fase G2 e picchi durante la mitosi. U2OS cellule che esprimono un FRET monitoraggio basato sonda Plk1 attività sono state girate 9,14 per 60 ore utilizzando un sistema Deltavision Spectris Imaging dotato di un NA 20x 0,7 obiettivo aria e una lampada al mercurio. Condizioni di imaging sono stati selezionati per causare fototossicità minimo, come indicato nella sezione 2, con binning 4x e filtri a densità neutra che bloccano il 99% della luce in entrata. Una rappresentazione in falsi colori della invertito FRET-rapporto, a seguito di una cella a 4 divisioni. B, chimografo della cellula mostrata in A dopo l&#39;applicazione di un filtro medio. C, la quantificazione del rapporto invertito FRET della cellula mostrata in A. D, ingresso mitotico cumulativo di 50 FRET-sonda cellule che esprimono, tra cui le divisioni di cellule figlie.

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Non abbiamo nulla da rivelare.

Monitoraggio FRET durante il ciclo cellulare richiede considerazioni che sono meno cruciali nella valutazione a breve termine le risposte agli stimoli esterni. In primo luogo, la progressione del ciclo cellulare è facilmente perturbato da segnalazione stress, richiedendo che fototossicità è ridotto al minimo. In secondo luogo, tutti i giornalisti possono potenzialmente influenzare processi cellulari per titolazione di fuori chinasi, fosfatasi o domini di interazione. Il modo forse più semplice per valutare se le con...

<table border="1"><tbody><tr><td> Reagente </td><td> Numero di catalogo </td><td> Azienda </td></tr><tr><td> Leibovitz L-15, senza rosso fenolo </td><td> 21083-027 </td><td> GIBCO, di Life Technologies </td></tr><tr><td> DMEM + Glutamax-I </td><td> 31966 </td><td> GIBCO, di Life Technologies </td></tr><tr><td> Siero fetale bovino (FBS) </td><td> SV30160.03 </td><td> Hyclone </td></tr><tr><td> 0,05% tripsina EDTA </td><td> SH30236.01 </td><td> Hyclone </td></tr><tr><td> Penicillina-Streptomicina </td><td> SV30010 </td><td> Hyclone </td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO, di Life Technologies </td></tr><tr><td> Puromicina </td><td> P8833 </td><td> Sigma-Aldrich </td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

Förster risonanza trasferimento di energia (FRET) a base di reporter 1 consentono la valutazione della chinasi endogene e delle attività della fosfatasi nelle cellule viventi. Tali sonde consistono tipicamente di varianti del CFP e YFP, è intervenuto da una sequenza phosphorylatable e una fosfo-dominio di legame. Su fosforilazione, la conformazione della sonda cambiamenti, che si traduce in una variazione della distanza o l&#39;orientamento tra CFP e YFP, portando ad un cambiamento di FRET efficienza (Fig. 1). Più sonde sono state pubblicate nell&#39;ultimo decennio, monitorando l&#39;equilibrio tra l&#39;attività di chinasi e fosfatasi molteplici, tra cui i giornalisti di PKA 2, PKB 3, PKC 4, PKD 5, 6 ERK, JNK 7, Cdk 18, Aurora B 9 e 9 Plk1 . Dato il design modulare, le sonde aggiuntive possono emergere nel futuro vicino a 10. Progressione attraverso il ciclo cellulare è affetto da stress signaling percorsi 11. In particolare, il ciclo cellulare è regolato in modo diverso durante l&#39;imperturbabile crescita rispetto a quando le cellule si stanno riprendendo dallo stress 12. Time-lapse imaging di cellule attraverso il ciclo cellulare richiede quindi particolare cautela. Questo diventa un problema soprattutto quando si impiegano immagini raziometrici, visto che due immagini con un elevato rapporto segnale-rumore sono tenuti ad interpretare correttamente i risultati. Raziometrico FRET imaging di cambiamenti del ciclo cellulare dipendente in attività di chinasi e fosfatasi sono prevalentemente stata limitata a sotto-sezioni del ciclo cellulare 8,9,13,14. Qui si discute un metodo per monitorare FRET-based utilizzando sonde di imaging raziometriche tutto il ciclo delle cellule umane. Il metodo si basa sul materiale che è disponibile per molti ricercatori nelle scienze della vita e non richiede conoscenze specialistiche di microscopia o di elaborazione delle immagini.

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

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

FRET basato giornalisti sono sempre più utilizzati per monitorare le attività di chinasi e fosfatasi in cellule vive. Qui si descrive un metodo su come utilizzare FRET-based reporter per valutare ciclo-dipendente delle cellule cambiamenti nella fosforilazione di destinazione.

Monitoraggio Attività chinasi e fosfatasi attraverso il ciclo cellulare da raziometrica FRET

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

monitoring-kinase-phosphatase-activities-through-cell-cycle

Research

JoVE Journal

Biology

14.2K Views.  Karolinska Institutet. FRET basato giornalisti sono sempre più utilizzati per monitorare le attività di chinasi e fosfatasi in cellule vive. Qui si descrive un metodo su come utilizzare FRET-based reporter per valutare ciclo-dipendente delle cellule cambiamenti nella fosforilazione di destinazione.

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

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

Disegno di sangue di ratti attraverso la vena safena e da puntura cardiaca

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Ricerca

Biologia

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.

Analisi della posizione ciclo cellulare nelle cellule di mammifero

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

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A lungo termine del test di tossicità letale con il crostaceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET Microscopia per Monitoraggio in tempo reale di segnalazione di eventi in cellule vive Uso Biosensori unimolecolare

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Studiare Looping DNA a singola molecola FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

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.

Sincronizzazione di<em&gt; Caulobacter crescentus</em&gt; Per Indagine del ciclo cellulare batterica

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 ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Approcci sperimentale per lo studio mitocondriale localizzazione e funzione di un nucleare di cellule ciclo chinasi, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

Conformazioni GPCR g di proteine-selettivi misurata utilizzando FRET sensori in un Fluorometer Assay Live Cell Suspension

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

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.

Combinazione di sincronizzazione cellulare mitotica e microscopia confocale ad alta risoluzione per studiare il ruolo delle proteine del ciclo cellulare multifunzione durante la mitosi

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 ...