We would like to thank the Children&#8217;s Hospital at Montefiore, Department of Pediatrics, for providing support for these studies. We would like to thank the National Alopecia Areata Foundation for providing financial support for conducting studies with C3H/HeJ engrafted mice.

Etica Dichiarazione: Tutti gli studi che coinvolgono animali devono essere approvate dal IACUC dell&#39;istituzione (per i dati che segue, i protocolli sono stati approvati dal Montefiore IACUC, protocollo # 11-6-240 e 13-7-100 #). Gli animali vengono forniti anestesia leggeri come indicato al solo scopo di tenerli fermi durante fotografia, non esistono procedure dolorose richieste per questo protocollo. 1. Acquisizione di fotografie <ol><li> Impostare fuoco e campo visivo per imager gel utilizzando carta con il testo stampato. Verificare uniformità della fonte di luce in tutta regione fotografato. Per garantire l&#39;uniformità, utilizzare un imager gel con un costruito in fonte di luce per la fotografia riflettente. Non utilizzare una fonte di luce transilluminazione, in quanto ciò creerebbe una silhouette dell&#39;animale, che non è adatto per ulteriori analisi in scala di grigi. NOTA: Questo metterà il mouse leggermente fuori fuoco, che fornisce media ottica in tutta la regione di interesse (ROI) e volontàridurre gli errori quantali molto piccoli (cioè &lt;10 pixel) regioni di interesse. Regioni più grandi d&#39;interesse non saranno interessati da questo piccolo fuoco di regolazione, né saranno influenzati dalle differenze nelle dimensioni dell&#39;animale. </li><li> Anestetizzare animali utilizzando ketamina (100 mg / ml) / xilazina (20 mg / ml) (2: 1), in quanto fornisce un rapido effetto anestesia e rapido recupero ed è ottimale per fotografare multipli animali. NOTA: L&#39;anestesia è confermata quando l&#39;animale è ancora sufficiente per permettere fotografia. Come animali in genere recuperano da questa luce anestesia entro 10-15 minuti, il veterinario non ha raccomandato l&#39;uso di pomate oculari. </li><li> Inserire animali anestetizzati su gel imager in allineamento verticale (più vicino a parallelo possibile). <ol><li> Per le fotografie dorsali, mettere gli animali in posizione prona con gli arti estesi. </li><li> Per le fotografie ventrali, mettere gli animali in posizione supina, facendo attenzione che gli animali non sono ruotati lateralmente. </li&gt; </ol></li><li> Luogo di serie in scala di grigi nella regione fotografato. </li><li> Chiudere la porta di accesso. Importante: luce ambientale può introdurre variazioni di esposizione. </li><li> Set F-stop di un&#39;esposizione che pone la regione di interesse all&#39;interno della gamma lineare di acquisizione (lettura F-stop). NOTA: La maggior parte dei sistemi saranno visualizzare in cui l&#39;immagine è satura. </li><li> Prendere fotografia. </li><li> Change F-Stop un&#39;altra impostazione dell&#39;esposizione che pone la regione di interesse all&#39;interno del range lineare aumentando o diminuendo il F-stop da 1, tale che sia il livello e la regione di interesse rimangono in una serie lineare di acquisizione (domanda F- stop). </li><li> Prendere fotografia. </li><li> Una volta che la fotografia è completata, posizionare gli animali su un tavolo di riscaldamento e monitorare fino a che non possono mantenere decubito sternale. Animali tornare alle loro gabbie. Restituisce il gruppo di animali di vivaio una volta che tutti gli animali sono completamente recuperati. </li></ol> 2. Quantificazione di assorbimento della luce &lt;/ P&gt; <ol><li> Regioni Mark di interesse sulle immagini degli animali uso del software fornito per il gel imager. <ol><li> Per tutta la vista dorsale animale, utilizzare un&#39;immagine rettangolare o ovale estende dalle arti superiori agli arti inferiori, che si estende lateralmente per quanto possibile tale che nessuna parte della scatola si estende oltre la parte posteriore dell&#39;animale come mostrato nella Figura 1A. NOTA: Si potrebbe anche segnare l&#39;area di interesse utilizzando uno strumento di disegno a mano libera. </li><li> Per animale intero vista ventrale, utilizzare 2 rettangoli: coprire la regione pelvica e coprire la zona del torace come mostrato nella Figura 1B. </li><li> Per una regione più piccola di interesse, vale a dire, la posizione di somministrazione del farmaco, marchio a seconda dei casi. </li></ol></li><li> Regione Mark (s) di interesse sullo standard di assorbimento in scala di grigi. </li><li> Assorbimento Registrare da ciascuna delle regioni marcate di interesse. </li></ol> 3. Analisi Livelli di assorbimento richiederlod per le regioni di interesse potrebbe essere necessario normalizzato allo standard di sfondo per il confronto tra le fotografie. Il rapporto di esposizione ad assorbimento è log-log (cioè, il rapporto è lineare tra log (esposizione) e di registro (assorbimento)). Con questo rapporto, l&#39;assorbimento della regione di interesse può essere normalizzato ad un valore sfondo standard, che permetterà assorbimenti di essere confrontati direttamente tra diverse fotografie, comprese quelle prese a diversi momenti (ad esempio, le misurazioni seriali all&#39;interno di un protocollo di studio). La procedura per l&#39;esecuzione di queste correzioni è dettagliata in passaggi facoltativi 3.1 e 3.2. <ol><li> (Opzionale) curva Aree log (esposizione) vs. log (assorbimento) con valori ottenuti dallo standard di assorbimento in scala di grigi. </li><li> (Opzionale) Regolare le variazioni di livello di ogni fotografia come segue: <ol><li> Selezionare F-stop per la lettura (come determinato al punto 1.6) e F-stop di riferimento (come determinato in sTep 1.8). </li><li> Calcolare assorbimento medio dello standard in tutte le fotografie alla lettura F-stop. Questa media è il valore di riferimento standard (RSV). </li><li> Calcolare differenza di assorbimento tra la lettura e le impostazioni di F-stop di riferimento in ogni fotografia per lo standard (delta-S) e per ogni ROI definita (delta-ROI-1, delta-ROI-2 ... ..delta-ROI-X) </li><li> Calcolare l&#39;assorbimento corretto per ogni ROI come segue: assorbimento corretto (ROI-X) = Assorbimento (ROI-X) a lettura F-stop + (RSV - assorbimento di serie a leggere F-stop) * (delta-ROI-X / delta-S) </li></ol></li><li> Compilare i dati sperimentali ed eseguire l&#39;analisi dei dati utilizzando tecniche statistiche standard per le variabili continue (ad esempio, ANOVA, T-test). </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-aided+quantification+of+scalp+hair.">Computer-aided quantification of scalp hair.</a> Dermatol Clin. 4 (4), 627-640 (1986).</li><li>Fleming, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+a+structural+analysis+of+dermatoscopic+imagery.">Techniques for a structural analysis of dermatoscopic imagery.</a> Comput Med Imaging Graph. 22 (5), 375-389 (1998).</li><li>Chamberlain, A. J., Dawber, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+evaluating+hair+growth.">Methods of evaluating hair growth.</a> Australas J Dermatol. 44 (1), 10-18 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Kodak/Tiffen Q-13 Gray Scale*. , .</li><li>Freyschmidt-Paul, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+alopecia+areata+in+C3H/HeJ+mice+with+the+topical+immunosuppressant+FK506+(Tacrolimus).">Treatment of alopecia areata in C3H/HeJ mice with the topical immunosuppressant FK506 (Tacrolimus).</a> Eur J Dermatol. 11 (5), 405-409 (2001).</li><li>Katikaneni, R., Ponnapakkam, T., Matsushita, O., Sakon, J., Gensure, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+and+prevention+of+chemotherapy-induced+alopecia+with+PTH-CBD,+a+collagen-targeted+parathyroid+hormone+analog,+in+a+non-depilated+mouse+model.">Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model.</a> Anticancer Drugs. 25 (1), 30-38 (2014).</li><li>Peters, E. 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Authors have nothing to disclose.

In questo rapporto una descrizione dettagliata è dotata di una nuova tecnica per quantificare la perdita dei capelli nei roditori. Questa tecnica utilizza un imager gel per l&#39;acquisizione delle immagini e analisi, apparecchi che è prontamente disponibile in molti laboratori. Le misurazioni hanno dimostrato di essere robusto per piccole variazioni nella tecnica (figure 4 - 5), e sono ben correlati al grado di perdita di capelli visiva in modelli di trattamento (Figura 6).</...

Alopecia (perdita dei capelli) può essere un evento psicologicamente ed emotivamente angosciante con molteplici cause. Maschio-calvizie è la causa più comune di alopecia, che colpisce circa due terzi dei maschi per età 35 1. Un modello simile di perdita dei capelli può essere osservato nelle donne con sindrome dell&#39;ovaio policistico. In entrambi questi disturbi, la perdita di capelli è mediata androgeni. Alopecia può verificarsi anche come una malattia autoimmune, alopecia areata, che colpisce 1,7% della popolazione 2. Alopecia può verificarsi come un effetto collaterale di alcuni trattamenti medici, come la chemioterapia 3. Una percentuale elevata (65-85%) dei pazienti in chemioterapia esperienza un certo grado di alopecia 4,5. Le conseguenze psicologiche della perdita di capelli sono stati ben studiati in ambito chemioterapia. Alopecia indotta da chemioterapia può causare ansia, depressione, un&#39;immagine negativa del corpo, abbassato l&#39;autostima e una riduzione del senso di benessere 6,7. Un alto percentuale (47-58%) dei pazienti oncologici femminili considerare la perdita di capelli per essere l&#39;aspetto più traumatico della chemioterapia, e fino a 8% trattamento calo per paura di perdita di capelli 4,6. Ci sono anche prove in alopecia androgenetica per sostenere la terapia per ridurre le conseguenze psicologiche e anche medici di perdita di capelli 8,9. Allo stesso modo, l&#39;alopecia areata è stato segnalato per avere gravi conseguenze psicologiche 2, e la natura irregolare della perdita dei capelli può portare a un risultato estetico più sgradevole rispetto alla maggior parte altre cause di perdita dei capelli. Mentre i farmaci con effetti anti-androgenici lieve (cioè, spironolattone) erano stati utilizzati con successo limitato come terapia per l&#39;alopecia, il primo farmaco efficace per l&#39;alopecia era minoxidil 10. Questo antipertensivo ha un osservato effetto collaterale di causare la crescita dei capelli, ed è ora utilizzato come terapia topica per molte forme di alopecia. Tuttavia, le risposte sono spesso incomplete, con alcuni soggetti che mostra solo lentoing di perdita di capelli, piuttosto che la ricrescita reale 10. Finasteride è un antagonista competitivo di tipo II 5α-reduttasi che blocca la conversione del testosterone in diidrotestosterone, con conseguente miglioramento della alopecia androgenetica a scapito di parziale androgeni blocco sistemico. I tassi di risposta con-lungo termine (10 anni) terapia sono circa il 50% 11. Nel complesso, nonostante una notevole ricerca in questo settore, non c&#39;è ancora una terapia adeguata per la perdita dei capelli. Per decenni, gli scienziati e medici hanno esaminato i metodi di misurazione della crescita dei capelli del cuoio capelluto in studi clinici. Con lo sviluppo di farmaci che trattano alopecia, vi è stata una maggiore necessità di mezzo affidabile, economico e minimamente invasivi per misurare la crescita dei capelli e, in particolare, la risposta alla terapia. Tecnologia di analisi di immagine per una quantificazione precisa della densità dei capelli in pazienti con disturbi di perdita dei capelli ha dato risultati coerenti e validi in passato utilizzando una varietà di techniques, compresa l&#39;analisi delle immagini digitalizzate 12, l&#39;analisi delle immagini di singoli peli e lesioni 13 della pelle, e la scansione microscopica per quantificare la massa di capelli in una regione del cuoio capelluto definita 14. Purtroppo, mentre le metodologie di cui sopra hanno fornito una migliore valutazione dell&#39;efficacia per i capelli la crescita-promuovere interventi in studi clinici, questi metodi non siano state applicate a studi su roditori in studi preclinici. Il nostro obiettivo è quello di sviluppare un metodo coerente e affidabile per quantificare la perdita dei capelli nei topi, che permetterà ai ricercatori di valutare con maggiore precisione e confrontare nuovi approcci terapeutici per varie forme di alopecia. Abbiamo sviluppato una metodologia utilizzando attrezzature facilmente reperibili nella maggior parte dei laboratori che permetteranno la quantificazione rapida e affidabile della densità dei capelli nei topi con capelli castani o neri. Questa metodologia è stata testata in modelli murini di chemioterapia-indotta alopecia, alopecia areata, e alopecia da waxing. Un protocollo dettagliato è presentato per l&#39;esecuzione di queste misure, compresi i dati di convalida da C57BL / 6 e C3H / ceppi HeJ di topi. Poiché questa tecnica si basa sulla rilevazione di assorbimento della luce da pigmenti nel capello, non può essere utilizzato per rilevare la crescita dei capelli in topi bianchi o topi albini.

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">KODAK Gel Logic 100 Imaging System&#160;&#160;</td>
			<td style="width:121px;">Eastman Kodak Company, Rochester, NY, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gel imager for obtaining and analyzing photographs, must have built in light source for reflective photography.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">The Kodak/Tiffen Q-13 Gray Scale</td>
			<td style="width:121px;">Imatest, http://www.imatest.com/docs/q13/</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Greyscale standard</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C57BL/6J Mice</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">C3H/HeJ engrafted mouse</td>
			<td style="width:121px;">&#160;Jackson Laboratories, Bar Harbor, Maine</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Mice for representative study</td>
		</tr>
	</tbody>
</table>

a new technique for quantitative analysis of hair loss in mice using grayscale analysis

Alopecia è una forma comune di perdita dei capelli che può verificarsi in diverse condizioni, tra cui la perdita di capelli maschile-modello, la sindrome dell&#39;ovaio policistico, e alopecia areata. Alopecia può verificarsi anche come effetto collaterale della chemioterapia in pazienti affetti da cancro. In questo studio, il nostro obiettivo era quello di sviluppare un metodo coerente e affidabile per quantificare la perdita dei capelli nei topi, che permetterà agli investigatori di valutare con precisione e confrontare nuovi approcci terapeutici per queste varie forme di alopecia. Il metodo utilizza un imager gel standard per ottenere ed elaborare immagini di topi, misurando l&#39;assorbimento di luce, che si verifica in proporzione agitato per la quantità di capelli nero (o grigio) sul mouse. Dati che sono stati quantificati in questo modo possono essere analizzati con tecniche statistiche standard (ad esempio, ANOVA, T-test). Questa metodologia è stata testata in modelli murini di chemioterapia-indotta alopecia, alopecia areata e alopecia da ceretta. In questo rapporto, il protocollo dettagliato è presENTED per l&#39;esecuzione di queste misure, compresi i dati di convalida da C57BL / 6 e C3H / ceppi HeJ di topi. Questa nuova tecnica offre una serie di vantaggi, tra cui relativa semplicità di applicazione, dipendenza apparecchiature che è prontamente disponibile nella maggior parte dei laboratori di ricerca, e l&#39;applicazione di un obiettivo, valutazione quantitativa che è più robusto di valutazioni soggettive. Miglioramenti nella quantificazione della crescita dei capelli nei topi miglioreranno studio di modelli alopecia e facilitare la valutazione di promettenti nuove terapie in studi preclinici.

Questa tecnica è stata convalidata utilizzando C3H / HeJ trapiantate, il modello murino di alopecia areata 16. Questi animali sviluppano la perdita di capelli globale che progredisce gradualmente nel tempo. Tuttavia, la perdita dei capelli avviene a chiazze, e può variare da un mouse al successivo, introducendo notevole variabilità e ostacolando valutazioni qualitative. Questo modello ha fornito l&#39;occasione per testare le correlazioni tra misurazioni della densità ottica in diversi punti il ​​mous...

Watch this Scientific Journal Video about A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis at JoVE.com

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Una nuova tecnica di analisi quantitativa di perdita dei capelli nei topi mediante analisi in scala di grigi

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

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8.9K Views.  Children's Hospital at Montefiore. Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Video: A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Ricerca

Biologia

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-cell imaging e analisi quantitativa della embrionali in cellule epiteliali Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

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 ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Analisi quantitativa della migrazione casuale di celle usando Time-lapse Microscopia Video

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

A Quantitative Analysis Fitness Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Rapida analisi genetica di epitelio-mesenchimale segnalazione durante la rigenerazione dei capelli

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Analisi quantitativa di autofagia usando 3D avanzato microscopia a fluorescenza

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Un nuovo approccio per l&#39;analisi comparativa dei multiproteico Complessi Basato su<sup&gt; 15</sup&gt; N metabolica Etichettatura e quantitativa Spettrometria di Massa

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Un saggio di legame High Throughput MHC II per l&#39;analisi quantitativa dei peptidi epitopi

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...