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.

Ethik Hinweise: Alle Studien, die Tiere müssen von der IACUC der Einrichtung genehmigt werden (für die folgenden Daten wurden Protokolle durch die Montefiore IACUC, Protokoll # 11-6-240 und 13-7-100 # zugelassen). Tiere sind vorhanden Licht Anästhesie für den alleinigen Zweck halten sie noch während der Aufnahme angezeigt wird, gibt es keine schmerzhaften Eingriffen für dieses Protokoll erforderlich. 1. Erwerb von Fotografien <ol><li> Stellen Sie Fokus und Sichtfeld für Gel-Imager Papier mit gedrucktem Text. Stellen Sie sicher, Gleichmäßigkeit der Lichtquelle über fotografiert Region. Um sicherzustellen, Einheitlichkeit, mit einem Gel-Imager mit in Lichtquelle für reflektierende photography eine gebaut. Verwenden Sie keinen Durchlichtquelle, da dies eine Silhouette des Tieres, die für die weitere Analyse ungeeignet ist Graustufen erstellen. Hinweis: Dadurch wird die Maus leicht unscharf, die optische Mittel in der Region of Interest (ROI) bietet Platz und wirdHilfe Quanten Fehler zu reduzieren für sehr kleine (dh &lt;10 Pixel) Bereiche von Interesse. Größere Bereiche von Interesse werden durch diese geringfügige Fokussierungseinstellung beeinträchtigt werden, noch werden sie durch Unterschiede in der Größe des Tieres berührt. </li><li> Anesthetize Tiere unter Verwendung von Ketamin (100 mg / ml) / Xylazin (20 mg / ml) (2: 1), da dies eine schnelle Anästhesiewirkung und eine schnelle Wiederherstellung und ist optimal für das Fotografieren mehrerer Tiere. HINWEIS: Anesthesia wird bestätigt, wenn das Tier noch genug Fotografie ermöglichen. Da die Tiere in der Regel von dieser Licht Anästhesie innerhalb von 10-15 Minuten zu erholen, hat der Tierarzt nicht empfohlen Nutzung von Augensalben. </li><li> Zeigen narkotisierten Tiere auf Gel-Imager in vertikaler Ausrichtung (so nah wie möglich parallel). <ol><li> Für Rücken Fotografien, legen Sie die Tiere in Bauchlage mit Gliedmaßen erweitert. </li><li> Für ventralen Fotografien, platzieren Tiere in Rückenlage, wobei darauf geachtet, dass die Tiere nicht seitlich gedreht. </li&gt; </ol></li><li> Zeigen Graustufen-Standard in fotografiert Region. </li><li> Schließen Zugangstür. Wichtig: Umgebungslicht kann Schwankungen Exposition einzuführen. </li><li> Stellen Sie Blende zu einer Exposition, die den interessierenden Bereich im linearen Bereich des Erwerbs setzt (Lesen F-Stop). HINWEIS: Die meisten Systeme werden angezeigt, wo das Bild ist gesättigt. </li><li> Nehmen Sie Fotos. </li><li> Ändern F-Stopp in einen anderen Belichtungseinstellung, die den interessierenden Bereich im linearen Bereich legt durch Erhöhung oder Verringerung der F-stop um 1, so dass sowohl die Standard- und die Region von Interesse in einer linearen Reihe von Erfassung (Referenz bleiben F- hör auf). </li><li> Nehmen Sie Fotos. </li><li> Sobald Fotografie abgeschlossen ist, legen Sie Tiere auf einem Tisch Erwärmung und zu überwachen, bis sie Brustlage halten kann. Zurück Tiere in ihre Käfige. Bringen Sie die Gruppe von Tieren auf das Vivarium, sobald alle Tiere vollständig erholt. </li></ol> 2. Quantifizierung der Lichtabsorption &lt;/ P&gt; <ol><li> Mark Regionen von Interesse auf die Bilder der Tiere unter Verwendung mitgelieferte Software für die Gel-Imager. <ol><li> Für Ganztier Dorsalansicht verwenden einen rechteckigen oder ovalen Bild sich von dem oberen Schenkel an den unteren Extremitäten, die sich seitlich so weit wie möglich, so dass kein Teil der Schachtel erstreckt sich über den Rücken des Tieres, wie in 1A gezeigt. HINWEIS: Man könnte auch markieren den Bereich von Interesse mit einem Freihand-Zeichenwerkzeug. </li><li> Für ganze Tier Ventralansicht Verwenden 2 Rechtecke: eine für den Beckenbereich und eine für den Brustbereich, wie in 1B gezeigt. </li><li> Für eine kleinere Region von Interesse, dh, die Lage der Arzneimittelverabreichung, Marke entsprechend. </li></ol></li><li> Region Marke (n) von Interesse auf Graustufen Absorption Standard. </li><li> Record Absorption von jeder der markierten Regionen von Interesse. </li></ol> 3. Analyse Absorptionsniveaus obtained für die Regionen von Interesse können müssen, um den Hintergrund-Standard für den Vergleich zwischen Fotografien normalisiert werden. Die Beziehung der Belastung durch Absorption log-log (dh die Beziehung linear zwischen log (Exposition) und log (Absorption)). Unter Verwendung dieser Beziehung kann die Absorption der interessierenden Region normiert auf eine Standard-Hintergrundwert, der ermöglicht Absorptionen direkt zwischen verschiedenen Aufnahmen, einschließlich derjenigen, die zu verschiedenen Zeitpunkten (dh seriellen Messungen innerhalb eines Studienprotokoll) aufgenommen verglichen werden. Das Verfahren für die Durchführung dieser Korrekturen wird in optionalen Schritte 3.1 und 3.2 beschrieben. <ol><li> (Optional) Plot Kurve von log (Belichtung) gegen log (Absorption) mit Hilfe von Graustufen Absorption Standard erhaltenen Werte. </li><li> (Optional) Stellen Sie Schwankungen in der Standard jedes Fotos, wie folgt: <ol><li> Wählen Sie F-Stop für das Lesen und F-Stop Referenz (wie in Schritt 1.6 festgelegt) (wie in s bestimmttep 1.8). </li><li> Berechnen Sie die durchschnittliche Absorption der Standard in allen Aufnahmen am Lesen F-stop. Dieser Durchschnitt ist die Referenz-Standard-Wert (RSV). </li><li> Berechnen Absorptionsdifferenz zwischen dem Lesen und Referenzblendeneinstellungen in jeder Photographie für den Standard (Delta-S) und für jeden definierten ROI (delta-ROI-1, delta-ROI-2 ... ..delta-ROI-X) </li><li> Berechnen Sie die korrigierte Absorptions für jede ROI wie folgt: Korrigierte Absorption (ROI-X) = Absorption (ROI-X) im Lesen F-stop + (RSV - Aufnahme von Standard zu lesen F-stop) * (delta-ROI-X / delta-S) </li></ol></li><li> Kompilieren experimentellen Daten und eine Datenanalyse unter Verwendung von statistischen Standardtechniken für kontinuierliche Variablen (das heißt, ANOVA, T-Test). </li></ol>

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Authors have nothing to disclose.

In diesem Bericht wird eine detaillierte Beschreibung einer neuen Technik zur Quantifizierung von Haarausfall bei Nagetieren ist. Diese Technik verwendet ein Gel Imager für Bilderfassung und -analyse, die Ausrüstung, die in den meisten Laboratorien leicht verfügbar ist. Die Messungen haben gezeigt, robuste auf geringfügige Änderungen in der Technik (Figuren 4 - 5) zu sein, und mit dem Grad der visuellen Haarausfall bei Behandlungsmodelle (Abbildung 6) gut korrelier...

Alopezie (Haarausfall) kann eine psychologisch und emotional belastenden Ereignis mit mehreren Ursachen. GPH ist die häufigste Ursache von Haarausfall, die etwa zwei Drittel der Männer im Alter von 35 ein. Ein ähnliches Muster des Haarausfalls kann bei Frauen mit PCO-Syndrom beobachtet werden. In beiden dieser Erkrankungen wird der Haarausfall Androgenrezeptor vermittelten. Alopecia können auch als Autoimmunkrankheit, Alopezie areata, die 1,7% der Bevölkerung betroffen 2 auftreten. Alopezie kann als Nebeneffekt von einigen medizinischen Behandlungen, wie die Chemotherapie 3 auftreten. Ein hoher Prozentsatz (65-85%) der Chemotherapie-Patienten erleben ein gewisses Maß an Haarausfall 4,5. Die psychischen Folgen von Haarausfall sind gut in der Chemotherapie-Einstellung untersucht. Chemotherapie-induzierter Alopezie kann in Angst, Depression, ein negatives Körperbild, senkte das Selbstwertgefühl und einer verringerten Gefühl von Wohlbefinden 6,7 führen. Eine hohe percentage (47-58%) der weiblichen Krebspatienten überlegen Haarausfall um die traumatischen Aspekt der Chemotherapie sein und bis zu 8% Rückgang Behandlung aus Angst vor Haarausfall 4,6. Es gibt auch Hinweise in androgenetische Alopezie, die Therapie, psychologische und sogar medizinischen Folgen von Haarausfall 8,9 reduzieren unterstützen. Ebenso Alopecia areata wurde berichtet, dass schwere psychische Folgen 2 haben und die lückenhafte Natur der Haarausfall kann zu einer unangenehmen kosmetische Ergebnis als die meisten anderen Ursachen für Haarausfall führen. Während Medikamente mit milden anti-androgenen Wirkungen (dh Spironolacton) hatten mit mäßigem Erfolg als Therapie für Alopezie verwendet wurde, das erste wirksame Medikament für Alopezie Minoxidil war 10. Diese antihypertensive hat eine beobachtete Nebenwirkung verursacht Haarwachstum, und wird nun als topische Therapie für viele Formen der Alopezie verwendet. Allerdings sind Reaktionen oft unvollständig, mit einigen Themen, die nur langsaming von Haarausfall statt tatsächlichen Nachwachsen 10. Finasterid ist ein kompetitiver Antagonist II 5α-Reduktase zu Dihydrotestosteron Typ blockier Umwandlung von Testosteron, was zu Verbesserungen in der androgenen Alopezie zu Lasten der Teil systemische Androgenblockade. Die Ansprechraten mit langfristigen (10 Jahre) Therapie sind etwa 50% 11. Insgesamt trotz beträchtlicher Forschung in diesem Bereich gibt es immer noch keine angemessene Therapie für Haarausfall. Seit Jahrzehnten haben Forscher und Kliniker Methoden zur Messung der Kopfhaarwachstum in klinischen Studien untersucht. Mit der Entwicklung von Medikamenten, die Alopezie zu behandeln, hat es einen erhöhten Bedarf an zuverlässigen, wirtschaftlichen und minimal invasive Mittel zum Messen des Haarwuchses und insbesondere als Reaktion auf die Therapie. Bildanalysetechnik für eine genaue Quantifizierung der Haardichte bei Patienten mit Haarausfall Erkrankungen ergab konsistent und gültige Ergebnisse in der Vergangenheit unter Verwendung einer Vielzahl von techniques, einschließlich der Analyse der digitalisierten Bilder 12, Bildanalyse der einzelnen Haare und Hautläsionen 13 und mikroskopische Scannen in Haarmasse in einem definierten Kopfbereich 14 zu quantifizieren. Leider, während die oben genannten Methoden haben verbesserte Beurteilung der Wirksamkeit für das Haarwachstum fördernden Eingriffe in klinischen Versuchen, in diesen Verfahren nicht auf Studien an Nagetieren in präklinischen Untersuchungen angewendet. Unser Ziel ist es, eine konsistente und zuverlässige Methode, um Haarausfall bei Mäusen, die es erlauben Ermittler genauer bewerten und zu vergleichen neue Therapieansätze für verschiedene Formen von Haarausfall wird quantifizieren zu entwickeln. Wir haben eine Methodik mit Hilfe von Geräten in den meisten Labors, die eine schnelle und zuverlässige Quantifizierung der Haardichte bei Mäusen mit braunen oder schwarzen Haares ermöglicht leicht verfügbar entwickelt. Diese Methode wurde in Maus-Modellen von chemotherapieinduzierter Alopezie, Alopecia areata und Alopecia von Waxin getestetg. Ein detailliertes Protokoll zur Durchführung dieser Messungen, einschließlich der Validierung Daten von C57BL / 6 und C3H / HeJ-Mäusestämme dargestellt. Da dieses Verfahren beruht auf der Erfassung der Lichtabsorption von Pigmenten in den Haarschaft, kann es nicht verwendet werden, um das Haarwachstum bei weißen Mäusen oder Albino Mäuse nachzuweisen.

<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

Alopezie ist eine häufige Form des Haarausfalls, die in vielen verschiedenen Bedingungen, einschließlich der männlichen Haarausfall, Syndrom der polyzystischen Ovarien und Alopecia areata auftreten können. Alopezie kann auch als Nebenwirkung der Chemotherapie bei Krebspatienten auftreten. In dieser Studie war es unser Ziel, eine konsistente und zuverlässige Methode, um Haarausfall bei Mäusen, die es erlauben Forschern, genau zu beurteilen und vergleichen Sie neue Therapieansätze für diese verschiedenen Formen von Haarausfall wird quantifizieren zu entwickeln. Das Verfahren nutzt ein Gel Standardbildgeber, um Bilder von Mäusen erhalten und verarbeiten, die Messung der Lichtabsorption, die in groben Verhältnis zu der Menge von Schwarz (oder Grau) Haar auf der Maus erfolgt. Daten, die auf diese Weise quantitativ bestimmt worden ist, kann dann unter Verwendung von statistischen Standardtechniken (dh, ANOVA, t-Test) analysiert. Diese Methode wurde in Mausmodellen von Chemotherapie-induzierter Alopezie, Alopecia areata und Alopecia aus Wachsen getestet. In diesem Bericht ist die detaillierte Protokoll prestierten zur Durchführung dieser Messungen, einschließlich der Validierung Daten von C57BL / 6 und C3H / HeJ-Mausstämme. Dieses neue Verfahren bietet eine Reihe von Vorteilen auf, einschließlich der relativen Einfachheit der Anwendung, der Verlässlichkeit von Geräten, die in den meisten Laboratorien leicht verfügbar ist, und Anlegen eines objektive, quantitative Beurteilung, die robuster als die subjektiven Bewertungen ist. Verbesserungen bei der Quantifizierung von Haarwachstum bei Mäusen wird Studie von Alopecia Modelle zu verbessern und Auswertung der vielversprechende neue Therapien in präklinischen Studien.

Diese Technik wurde mit C3H / HeJ Mäusen eingepflanzt, die Maus-Modell der Alopecia areata 16 validiert. Diese Tiere entwickeln globale Haarausfall die nach und nach im Laufe der Zeit fortschreitet. Allerdings tritt der Haarausfall bei Patches, und können von der Maus zum nächsten variieren, die Einführung erhebliche Variabilität und erschweren qualitative Bewertungen. Dieses Modell eine Gelegenheit, um Korrelationen zwischen den Messungen der optischen Dichte an unterschiedlichen Stellen auf der Maus, w...

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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.

Eine neue Technik zur quantitativen Analyse von Haarausfall in Mäuse mit Graustufen-Analyse

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 Aufrufe.  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.

Serie: 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.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

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

Forschung

Biologie

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 und Quantitative Analyse der embryonalen Epithelzellen in 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.

Quantitative Live Cell Fluoreszenz-Mikroskopie Analyse von 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 ...

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.

Quantitative Analyse der ungerichtete Migration von Zellen unter Verwendung Zeitraffer-Video-Mikroskopie

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.

Schnelle Genetische Analyse von epithelial-mesenchymale Signaling Während Haarregeneration

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.

Quantitative Analyse von Autophagie mit Advanced 3D Fluoreszenzmikroskopie

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.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung 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.

Ein neuer Ansatz für die vergleichende Analyse von Multiproteinkomplexen Basierend auf<sup&gt; 15</sup&gt; N Metabolische Markierung und quantitative Massenspektrometrie

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.

Ein hoher Durchsatz MHC II-Bindungsassay für die quantitative Analyse von Peptid-Epitope

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