Questo lavoro è stato sostenuto da NIH di Grant 1R21AG031965-01A1. MK è una Ellison Medical Foundation Scholar Nuove invecchiamento.

Parte 1: Preparazione delle colture invecchiamento <ol><li> Ceppi striscia di interesse da parte scorte congelate su piastre di agar YEPD (1 estratto di lievito%, 2% bacto-peptone, 2% agar, 2% di glucosio). </li><li> Incubare le cellule a 30 ˚ C per 48 ore o fino a singole colonie appaiono. </li><li> Scegli singole colonie e inoculare in 5 ml di liquido YEPD (1 estratto di lievito%, 2% bacto-peptone, 2% di glucosio) in provetta. </li><li> Grow culture notte a 30 ˚ C, mantenendo costante agitazione utilizzando uno shaker o Rullo. </li><li> Inoculare 5 ml di sintesi completa (SC), medio (Tabella 1) con 50 microlitri della cultura durante la notte. Generalmente tre culture SC sono preparati per ogni sforzo per assicurare la replica biologico triplice copia delle analisi ciclo di vita per ogni ceppo in esame. </li><li> Mantenere le culture a 30 ˚ C con agitazione costante su un rullo di tamburo per l&#39;intero esperimento (in genere 2 o più settimane). </li></ol> Parte 2: Facendo una vitalità età-punto Dopo due giorni di cultura in SC mezzi di comunicazione, le cellule devono essere in fase stazionaria e la prima età punto è pronto per essere preso. Successive età punti dovrebbero essere prese ogni 2-3 giorni per un minimo di due settimane. Per ogni punto di età: <ol><li> Preparare il Bioscreen piatti a nido d&#39;ape 100-bene per l&#39;inoculazione, compilando tutti i pozzetti con 145 ml di YEPD. Assicurarsi di lasciare almeno un pozzetto riempito solo YEPD e non le cellule per l&#39;analisi dei dati in seguito. </li><li> Rimuovere le culture invecchiamento dal termostato. </li><li> Mescolare brevemente nel vortex la prima cultura ad essere inoculato nella piastra a nido d&#39;ape, facendo attenzione a non versare della cultura. </li><li> Prelevare 5 ml di cultura mista e pipetta in primo pozzo della piastra a nido d&#39;ape. Fiamma la bocca di ogni provetta prima e dopo la rimozione dei 5 aliquota microlitri. </li><li> Ripetere questa procedura per ogni cultura invecchiamento avendo cura di notare la posizione e corrispondenti a ciascuna cultura. Posizioni identiche bene dovrebbe essere utilizzato per ogni successiva età-point durante l&#39;intero esperimento. </li><li> Sostituire le culture nel 30 ˚ C incubatore al termine con la vaccinazioni. </li></ol> Parte 3: Caricamento del Bioscreen C macchina MBR <ol><li> Esporre il vano incubatore sollevando il coperchio e rimuovere il coperchio per il vassoio campione. </li><li> Inserire la nuova piastra inoculata a nido d&#39;ape nel vassoio del campione (usare lo slot di sinistra se siete solo leggendo una piastra). </li><li> Sostituire il coperchio per il vassoio campione e abbassare il coperchio del vano incubatrice. </li><li> Verificare che il fluido termovettore è superiore al livello minimo di riempimento. Se basso, aggiungere più di 1000 con pipetta microlitri con il fluido termovettore in dotazione. </li><li> Utilizzando il software Bioscreen &quot;EZExperiment&quot;, impostare i seguenti parametri per ottenere curve di crescita adatto per Saccharomyces cerevisiae: <ul><li> Numero di campioni: Inserire il numero di pozzi con i media o 200 </li><li> Filtro: 420-580nm, a banda larga </li><li> Temperatura: 30 ˚ C </li><li> Esperimento Durata: 1 giorno, 0 ore, 0 secondi </li><li> Intervallo di misura: 30 minuti </li><li> Agitazione: On, scuotendo continuo, ad alta </li></ul></li><li> Fare clic su &quot;Start&quot; per iniziare la lettura. </li></ol> Parte 4: Analisi dei dati Solitamente, a sei anni-punti sono presi nel corso di due settimane. L&#39;età punti sono prese a giorni 2, 4, 6, 9, 11 e 13. A seconda del disegno sperimentale e le tensioni in fase di sperimentazione, può essere opportuno tenere età punti più o meno frequentemente o per più di 2 settimane. E &#39;importante caricare la piastra a nido d&#39;ape nello stesso ordine per ogni età-point in modo tale che ogni cultura invecchiamento corrisponde alla stessa posizione e ad ogni età-punto, perché in questo modo l&#39;analisi dei dati molto più facile. <ol><li> Ottenere i file di output dalla macchina Bioscreen C MBR. Il &quot;EZExperiment&quot; software visualizzerà i dati Bioscreen come un file delimitato da tabulazioni che è compatibile con Microsoft Excel e altri software. La prima colonna indica a che ora la misura OD è stata presa durante l&#39;esperimento, con le successive colonne rappresentano ciascun pozzetto nelle piastre Bioscreen a nido d&#39;ape (Figura 1). </li><li> Eliminare la prima lettura OD da ogni colonna. Questa lettura è &quot;rumore&quot;. </li><li> Normalizzare i dati sottraendo il valore OD del bene con YEPD solo da tutti i valori di OD in ogni colonna. Questo rimuove l&#39;assorbanza di fondo da parte dei media. </li><li> Tracciare la curva escrescenza. Le curve si sposterà in funzione dell&#39;età (Figura 2). Per esempio, tracciando le curve escrescenza dalla colonna 1 (e 1 del piatto a nido d&#39;ape) oltre i sei punti del tempo, c&#39;è uno spostamento verso destra distinta delle curve nel tempo. </li><li> Calcolare il tempo di raddoppio (δ) per ogni bene in base alla cinetica di crescita del giorno 2 età-point. L&#39;equazione utilizzata per calcolare il tempo di raddoppio is: <img src="/files/ftp_upload/1156/equat.png" alt="Equazione" /> dove OD OD 1 e 2 rappresentano successive misurazioni OD e t 1 e t 2 è il tempo tra le misurazioni. Calcola il raddoppio volte solo tra i valori OD di 0,2 a 0,5. La media di questi valori è il tempo di raddoppio per quel bene. Calcola il raddoppio volte solo tra i valori OD di 0,2 a 0,5. La media di questi valori è il tempo di raddoppio per quel bene. La maggior parte dei ceppi wild-type lievito dovrebbe dare un valore tempo di raddoppio tra 85 a 90 minuti. </li><li> Per ogni età-punto, calcolare il tempo di spostamento (At) nelle curve escrescenza relativi ai primi anni-punto (giorno 2). Un modo semplice per farlo è quello di determinare la differenza nella lunghezza del tempo impiegato per ogni bene per raggiungere un diametro di 0,3 tra il punto iniziale per età e ogni successiva età-point. Il tempo che un particolare così raggiunto un diametro di 0,3 può essere calcolato con l&#39;equazione di regressione lineare corrispondente alla ln (OD) in funzione del tempo tra le due parentesi OD tempo-punti = 0,3. </li><li> Calcolare la frazione di sopravvissuti ad ogni età-point al fine di generare una curva di sopravvivenza (Figura 3). Definire il punto iniziale per età (giorno 2) di essere al 100% vitalità. Per ogni successivo all&#39;età punto calcolare la percentuale di sopravvivenza con l&#39;equazione: <img src="/files/ftp_upload/1156/equat2.png" alt="Equazione secondo" width="245" height="115" /> dove s n è la percentuale di sopravvivenza, Dt n è il turno di tempo, e δ n è il tempo di raddoppio. </li><li> Generare le curve di sopravvivenza (Figura 3B) come desiderato per ciascun bene (o pozzi replica) tracciando la frazione (o percentuale) di cellule vitali in funzione dell&#39;età. </li><li> Calcolare la sopravvivenza integrale (SI) per ogni bene. SI è definita come l&#39;area sotto la curva di sopravvivenza e può essere stimato con la formula: <img src="/files/ftp_upload/1156/equat3.png" alt="Equazione 3" width="400" height="79" /> in cui l&#39;età è l&#39;età n-punto (per esempio 2, 4, 6, 9, 11 e 13) e s n è il valore di sopravvivenza a quell&#39;età-point. </li><li> Determinare parametri statistici di pozzi replicare dai dati SI e la sopravvivenza. Comuni parametri statistici di interesse: media, mediana e varianza per ogni set di replica biologico. Una t-test o analisi simile può essere utilizzato per il confronto a coppie di SI a fini sperimentali e gruppi di controllo. Può anche essere auspicabile per generare le curve di sopravvivenza, come descritto in 4.8 da media biologici replica. </li></ol> Parte 5: risultati Rappresentante Al termine dell&#39;esperimento, si dovrà tracciare la curva di sopravvivenza ed eseguito l&#39;analisi dei dati sufficienti per determinare il potenziale di invecchiamento cronologico per diversi ceppi diversi o condizioni. Se eseguita correttamente, la crescita curve ottenute dalla macchina Bioscreen C MBR dovrebbe essere simile a quelli mostrati nella Figura 2 e le curve di sopravvivenza risultante dovrebbe essere simile a quelli mostrati nella figura 3. In generale, wild-type cellule coltivate nelle condizioni descritte qui avrà una durata di vita media per ordine cronologico di 7 giorni. Sostanziale variazione nella sopravvivenza si osserva in diversi ceppi e sotto alcune condizioni, come la crescita in media del glucosio 0,05%, la sopravvivenza mediana può superare i 30 giorni. <img src="/files/ftp_upload/1156/1156fig1.png" alt="" /> Figura 1. Uscita dati dal Bioscreen programma &quot;EZExperiment&quot;. Una colonna visualizza l&#39;ora in cui è stata presa una lettura di assorbanza. Colonne successive rappresentano la pozzetti della piastra a nido d&#39;ape inoculati con cellule prelevate da culture invecchiamento. <img src="/files/ftp_upload/1156/1156fig2.png" alt="" /> Figura 2. Curve escrescenza da un singolo replicare biologico nel corso di un esperimento. C&#39;è uno spostamento distinto nelle curve nel tempo come le cellule nella cultura invecchiamento perdono vitalità. Il periodo di tempo tra il punto di tempo iniziale (giorno 2) e un punto di tempo successivi determina la vitalità a questa età particolare. <img src="/files/ftp_upload/1156/1156fig3a.png" alt="" /><img src="/files/ftp_upload/1156/1156fig3b.png" alt="" /> Figura 3. A) Una curva di sopravvivenza generato utilizzando i dati escrescenza dalla Figura 1. I 2 punti di giorno è impostato come punto di vitalità del 100%. B), curve di sopravvivenza finale di due ceppi testati nello stesso esperimento. Queste curve di sopravvivenza Viabilità rappresentano la media di tre biologici repliche per ogni ceppo. Le barre di errore rappresentano la deviazione standard all&#39;interno biologici replica. L&#39;area ombreggiata sotto la curva di sopravvivenza rappresenta la sopravvivenza integrale (SI) per il ceppo 1. <table border="1"><tbody><tr><td colspan="2"> Tabella 1. Sintetico di media definite utilizzate per cronologica Studi Invecchiamento (ceppo backgrou° BY4743) </td></tr><tr><td> Componente </td><td> Concentrazione (g / L) </td></tr><tr><td> D-glucosio </td><td> 20 </td></tr><tr><td> Lievito di base di azoto (-AA/AS) </td><td> 1,7 </td></tr><tr><td> (NH 4) 2 SO 4 </td><td> 5.0 </td></tr><tr><td> Adenina </td><td> 0,04 </td></tr><tr><td> L-Arginina </td><td> 0,02 </td></tr><tr><td> L-acido aspartico </td><td> 0,1 </td></tr><tr><td> L-acido glutammico </td><td> 0,1 </td></tr><tr><td> L-Istidina </td><td> 0,1 </td></tr><tr><td> L-Leucina </td><td> 0,3 </td></tr><tr><td> L-lisina </td><td> 0,03 </td></tr><tr><td> L-metionina </td><td> 0,02 </td></tr><tr><td> L-Fenilalanina </td><td> 0,05 </td></tr><tr><td> L-Serina </td><td> 0,375 </td></tr><tr><td> L-treonina </td><td> 0,2 </td></tr><tr><td> L-triptofano </td><td> 0,04 </td></tr><tr><td> L-tirosina </td><td> 0,03 </td></tr><tr><td> L-Valina </td><td> 0,15 </td></tr><tr><td> Uracile </td><td> 0,1 </td></tr></tbody></table> Nota: Questa ricetta spiega auxotrophies diploidi BY4743 ceppo. Auxotrophies aminoacido deve essere compensata con l&#39;aggiunta di una concentrazione 5X finale al terreno sintetico completo.

<ol>
	<li>Steinkraus, K. A., Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+aging+in+yeast:+the+means+to+the+end.">Replicative aging in yeast: the means to the end.</a> Annu Rev Cell Dev Biol. 24, 29 (2008).</li><li>Kaeberlein, M., Conn, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of models for human aging. , 109 (2006).</li><li>Fabrizio, P., Longo, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chronological+life+span+of+Saccharomyces+cerevisiae.">The chronological life span of Saccharomyces cerevisiae.</a> Aging Cell. 2 (2), 73 (2003).</li><li>Murakami, C. J., Burtner, C. R., Kennedy, B. K., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+quantitative+analysis+of+yeast+chronological+life+span.">A method for high-throughput quantitative analysis of yeast chronological life span.</a> J Gerontol A Biol Sci Med Sci. 63 (2), 113 (2008).</li><li>Piper, P. W., Harris, N. L., MacLean, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preadaptation+to+efficient+respiratory+maintenance+is+essential+both+for+maximal+longevity+and+the+retention+of+replicative+potential+in+chronologically+ageing+yeast.">Preadaptation to efficient respiratory maintenance is essential both for maximal longevity and the retention of replicative potential in chronologically ageing yeast.</a> Mech Ageing Dev. 127 (9), 733 (2006).</li><li>Fabrizio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+is+a+mediator+of+an+altruistic+aging+program+in+Saccharomyces+cerevisiae.">Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae.</a> J Cell Biol. 166 (7), 1055 (2004).</li></ol>

L&#39;high-throughput vita saggio arco cronologico qui descritto è un metodo efficace per quantificare il potenziale di invecchiamento di un gran numero di ceppi con elevata accuratezza e precisione. L&#39;anticipo principale di questo metodo rispetto ai metodi classici per stabilire la sopravvivenza contando unità formanti colonie (ad esempio vedi 3) è l&#39;uso di un dispositivo di lettura shaker / incubatore / piastra, come la macchina Bioscreen C MBR di ottenere alta risoluzione curve di crescita a ogn...

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quantifying yeast chronological life span by outgrowth of aged cells

Il lievito in erba<em&gt; Saccharomyces cerevisiae</em&gt; Ha dimostrato di essere un organismo modello importante nel campo della ricerca sull&#39;invecchiamento<sup&gt; 1</sup&gt;. La replicazione e la durata della vita cronologico sono due paradigmi stabiliti utilizzato per studiare l&#39;invecchiamento nel lievito. Invecchiamento replicativo è definito come il numero di cellule figlie una sola cellula madre lievito produce prima di senescenza, invecchiamento cronologico è definito dalla lunghezza delle cellule tempo può sopravvivere in un non-divisione, quiescenza stato simile<sup&gt; 2</sup&gt;. Abbiamo sviluppato un elevato throughput metodo per la misurazione quantitativa della durata cronologica. Questo metodo comporta l&#39;invecchiamento delle cellule in un mezzo definito sotto agitazione ea temperatura costante. Ad ogni età-point, una sotto-popolazione di cellule viene rimosso dalla cultura invecchiamento e inoculati in terreno di coltura ricco. Ad alta risoluzione curva di crescita è quindi ottenuto per questa sotto-popolazione di cellule invecchiate con una macchina Bioscreen C MBR. Un algoritmo viene poi applicata per determinare la percentuale relativa di cellule vitali in ogni sub-popolazione basata sulla cinetica di crescita ad ogni età-point. Questo metodo richiede meno tempo e risorse rispetto ad altri saggi cronologico durata della vita, pur mantenendo la riproducibilità e precisione. L&#39;high-throughput natura di questo test dovrebbe consentire schermi genetiche e chimiche su larga scala per identificare i modificatori longevità romanzo per ulteriori test in organismi più complessi.

Watch this Scientific Journal Video about Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells at JoVE.com

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

Invecchiamento cronologico di lievito si riferisce alla perdita di vitalità cellulare associato con il tempo in fase stazionaria. Qui si descrivono un elevato throughput metodo per determinare quantitativamente la durata della vita cronologico lievito.

Quantificare cronologica Span lievito Life di conseguenza di cellule invecchiate

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

quantifying-yeast-chronological-life-span-by-outgrowth-of-aged-cells

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JoVE Journal

Biology

16.5K Views.  University of Washington. Invecchiamento cronologico di lievito si riferisce alla perdita di vitalità cellulare associato con il tempo in fase stazionaria. Qui si descrivono un elevato throughput metodo per determinare quantitativamente la durata della vita cronologico lievito.

Video: Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Misura Caenorhabditis elegans Life Span su terreni solidi

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Ricerca

Biologia

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Durata pannello misura replicazione in lievito

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificazione dei mitocondri da cellule di lievito

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Misura Caenorhabditis elegans Life Span in 96 piastre per microtitolazione Bene

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativa vivo cellulare-microscopio a fluorescenza Analisi di lievito Fission

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

La quantificazione temporale di espressione MAPK indotta in cellule di lievito singole

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Generazione e Cultura Sangue escrescenza endoteliali cellule da sangue periferico umano

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Monitoraggio e quantificazione processi di sviluppo in<em&gt; C. elegans</em&gt; Utilizzo strumenti open-source

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.