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.

Déclaration éthique: Toutes les études portant sur des animaux doivent être approuvés par le IACUC de l&#39;institution (pour les données qui suivent, les protocoles ont été approuvés par le IACUC Montefiore, le protocole # 11-6-240 et 13-7-100 #). Les animaux sont fournis anesthésie légère comme indiqué dans le seul but de les garder encore pendant la photographie, il n&#39;y a pas de procédures douloureuses requises pour ce protocole. 1. Acquisition de Photographies <ol><li> Mise au point et le champ de vision pour imageur de gel en utilisant du papier avec du texte imprimé. Vérifiez uniformité de source de lumière à travers la région photographiée. Pour assurer l&#39;uniformité, utiliser un imageur de gel avec un construit en source de lumière pour la photographie réfléchissante. Ne pas utiliser une source de lumière de transillumination, car cela créerait une silhouette de l&#39;animal qui est impropre à une analyse plus approfondie en niveaux de gris. NOTE: Ceci placera la souris légèrement floue, qui fournit moyenne optique à travers la région d&#39;intérêt (ROI) et la volontéaider à réduire les erreurs quantiques pour les très petites régions (c.-à-&lt;10 pixels) d&#39;intérêt. Grandes régions d&#39;intérêt ne seront pas affectés par cette mineure réglage de la focalisation, ils ne seront pas affectées par les différences dans la taille de l&#39;animal. </li><li> Anesthésier des animaux à l&#39;aide de kétamine (100 mg / ml) / xylazine (20 mg / ml) (2: 1), cela fournit l&#39;effet de l&#39;anesthésie rapide et une récupération rapide et est optimal pour photographier plusieurs animaux. NOTE: L&#39;anesthésie est confirmé lorsque l&#39;animal est encore assez pour permettre la photographie. Comme les animaux récupèrent de cette lumière dans l&#39;anesthésie 10-15 min, le vétérinaire n&#39;a pas recommandé l&#39;utilisation de pommades ophtalmiques. </li><li> Placer sur les animaux anesthésiés imageur gel en alignement vertical (aussi près que possible parallèle). <ol><li> Pour les photographies dorsales, placer les animaux en décubitus ventral avec des membres étendus. </li><li> Pour les photographies ventrales, placer les animaux dans la position couchée, en prenant soin que les animaux ne sont pas pivoté latéralement. </li&gt; </ol></li><li> Placez norme niveaux de gris dans la région photographiée. </li><li> Fermer la porte d&#39;accès. Important: La lumière ambiante peut introduire des variations dans l&#39;exposition. </li><li> Réglez F-stop à une exposition qui place la région d&#39;intérêt au sein de la gamme linéaire de l&#39;acquisition (lecture F-stop). REMARQUE: La plupart des systèmes afficher où l&#39;image est saturée. </li><li> Prenez la photo. </li><li> Changer F-stop à un autre réglage de l&#39;exposition qui place la région d&#39;intérêt au sein de la gamme linéaire en augmentant ou en diminuant le F-stop par une, de sorte que tant la norme et la région d&#39;intérêt restent dans une plage linéaire d&#39;acquisition (référence F arrêtez). </li><li> Prenez la photo. </li><li> Une fois que la photographie est terminé, placer les animaux sur une table de réchauffement et de suivre jusqu&#39;à ce qu&#39;ils puissent maintenir décubitus sternal. Retour animaux dans leurs cages. Remettre le groupe d&#39;animaux le vivarium fois tous les animaux sont complètement rétablis. </li></ol> 2. Quantification de l&#39;absorption de lumière &lt;/ P&gt; <ol><li> Marquer les régions d&#39;intérêt sur les images des animaux avec le logiciel fourni pour le gel imageur. <ol><li> Pour toute vue dorsale de l&#39;animal, utiliser une image rectangulaire ou ovale se étendant à partir des membres supérieurs aux membres inférieurs, se étendant latéralement autant que possible de telle sorte qu&#39;aucune partie de la boîte se étend au-delà du dos de l&#39;animal, comme illustré sur la figure 1A. NOTE: On peut aussi marquer la zone d&#39;intérêt en utilisant un outil de dessin à main levée. </li><li> Pour animal entier vue ventrale, utiliser deux rectangles: l&#39;un couvrant la région pelvienne et une couvrant la région de la poitrine comme le montre la figure 1B. </li><li> Pour une plus petite région d&#39;intérêt, ce est à dire, l&#39;emplacement de l&#39;administration du médicament, marque le cas échéant. </li></ol></li><li> Mark région (s) d&#39;intérêt sur la norme d&#39;absorption en niveaux de gris. </li><li> l&#39;absorption d&#39;enregistrement de chacune des régions marquées d&#39;intérêt. </li></ol> 3. Analyse les niveaux d&#39;absorption obtained pour les régions d&#39;intérêt peut avoir besoin d&#39;être normalisée à la norme de référence pour la comparaison entre les photographies. La relation entre l&#39;exposition à l&#39;absorption est logarithmique (ce est à dire, la relation est linéaire entre log (exposition) et log (absorption)). En utilisant cette relation, l&#39;absorption de la région d&#39;intérêt peut être normalisée à une valeur de référence standard, ce qui permettra absorptions à être comparées directement entre les différentes photographies, y compris celles qui sont prises à des moments différents (par exemple, des mesures en série au sein d&#39;un protocole de l&#39;étude). La procédure pour effectuer ces corrections est détaillé dans des étapes facultatives 3.1 et 3.2. <ol><li> (Facultatif) courbe de Terrain de log (exposition) en fonction de log (absorption) en utilisant les valeurs obtenues à partir de la norme d&#39;absorption en niveaux de gris. </li><li> (Facultatif) Réglez les variations dans le niveau de chaque photographie comme suit: <ol><li> Sélectionnez F-stop pour la lecture (tel que déterminé dans l&#39;étape 1.6) et F-stop pour référence (tel que déterminé à sTep 1,8). </li><li> Calculer absorption moyenne de la norme dans toutes les photographies à la lecture F-stop. Cette moyenne est la Valeur de référence standard (RSV). </li><li> Calculer différence d&#39;absorption entre la lecture et de référence réglages F-stop dans chaque photographie pour la norme (delta-S) et pour chaque ROI définie (delta-ROI-1, delta-ROI-2 ... ..delta-ROI-X) </li><li> Calculer l&#39;absorption corrigé pour chaque ROI comme suit: absorption corrigé (ROI-X) = Absorption (ROI-X) pour lire les F-stop + (RSV - absorption de la norme à lecture F-stop) * (delta-ROI-X / delta-S) </li></ol></li><li> Compiler les données expérimentales et effectuer des analyses de données en utilisant des techniques statistiques standard pour les variables continues (c.-à-ANOVA, T-test). </li></ol>

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Authors have nothing to disclose.

Dans ce rapport, une description détaillée est fournie d&#39;une nouvelle technique pour quantifier la perte de cheveux chez les rongeurs. Cette technique utilise un imageur gel d&#39;acquisition d&#39;images et d&#39;analyse, un équipement qui est facilement disponible dans la plupart des laboratoires. Les mesures ont été montrés pour être robuste aux variations mineures dans la technique (figures 4-5), et sont bien en corrélation avec le degré de perte de cheveux visuelle dans des modèles de...

Alopécie (perte de cheveux) peut être un événement psychologiquement et émotionnellement pénible aux causes multiples. Homme-calvitie est la cause la plus commune de l&#39;alopécie, affectant environ deux tiers des hommes de 35 ans 1. Une tendance similaire de la perte de cheveux peut être observée chez les femelles présentant un syndrome des ovaires polykystiques. Dans ces deux troubles, la perte de cheveux est médiée androgène. Alopécie peut également se produire comme une maladie auto-immune, la pelade, qui touche 1,7% de la population 2. L&#39;alopécie peut se produire comme un effet secondaire de certains traitements médicaux, tels que la chimiothérapie 3. Un pourcentage élevé (65-85%) des patients en chimiothérapie expérience dans une certaine mesure de l&#39;alopécie 4,5. Les conséquences psychologiques de la perte de cheveux ont été bien étudiés dans le cadre de la chimiothérapie. Alopécie induite par la chimiothérapie peut entraîner de l&#39;anxiété, la dépression, une image négative de leur corps, réduit l&#39;estime de soi et un sentiment réduite de bien-être 6,7. Une haute peourcentage (47-58%) des patients atteints de cancer chez les femmes considèrent la perte de cheveux à l&#39;aspect le plus traumatisant de la chimiothérapie, et jusqu&#39;à 8% de baisse de traitement par crainte de la perte de cheveux 4,6. Il existe également des preuves dans l&#39;alopécie androgénique pour soutenir thérapie pour réduire les conséquences psychologiques et même médicaux de perte de cheveux 8,9. De même, la pelade a été signalé à avoir des conséquences psychologiques graves 2, et de la nature inégale de la perte de cheveux peut conduire à un résultat esthétique plus désagréable que la plupart des autres causes de perte de cheveux. Alors que les médicaments ayant des effets anti-androgènes doux (ce est à dire, la spironolactone) avaient été utilisées avec un succès limité comme traitement pour l&#39;alopécie, le premier médicament efficace pour l&#39;alopécie était minoxidil 10. Ce antihypertenseur a un effet secondaire observé de provoquer la croissance des cheveux, et est maintenant utilisé comme traitement topique pour de nombreuses formes d&#39;alopécie. Cependant, les réponses sont souvent incomplets, certains sujets ne montrant que lentetion de la perte de cheveux plutôt que 10 repousse réel. Le finastéride est un antagoniste compétitif de taper II 5α-réductase qui bloque la conversion de testostérone en dihydrotestostérone, résultant en des améliorations dans l&#39;alopécie androgénique au détriment de blocage androgénique partielle systémique. Les taux de réponse avec à long terme (10 ans) thérapie sont environ 50% 11. Globalement, malgré des recherches considérables dans ce domaine, il n&#39;y a toujours pas de thérapie adéquate pour la perte de cheveux. Pendant des décennies, les scientifiques et les cliniciens ont examiné les méthodes de mesure de la croissance de cheveux de cuir chevelu dans les essais cliniques. Avec le développement de médicaments utilisés pour traiter l&#39;alopécie, on a un besoin accru de moyens fiables, économiques et peu invasive de mesure de la croissance des cheveux et, plus particulièrement, la réponse au traitement. Image en technologie d&#39;analyse pour une quantification précise de la densité des cheveux chez les patients présentant des troubles de perte de cheveux a abouti à des résultats cohérents et valables dans le passé en utilisant une variété de techniques, y compris l&#39;analyse des images numérisées 12, analyse de l&#39;image de poils individuels et les lésions 13 peau, et le balayage au microscope à quantifier la masse de cheveux dans une région du cuir chevelu 14 défini. Malheureusement, alors que les méthodes ci-dessus ont permis d&#39;améliorer l&#39;évaluation de l&#39;efficacité pour les cheveux favorisant la croissance interventions dans les essais cliniques, ces méthodes ne ont pas été appliquées aux études sur les rongeurs dans les études précliniques. Notre objectif est de développer une méthode cohérente et fiable de quantifier la perte de cheveux chez la souris, ce qui permettra aux enquêteurs d&#39;évaluer et de comparer de nouvelles approches thérapeutiques pour diverses formes d&#39;alopécie plus de précision. Nous avons développé une méthodologie utilisant des équipements facilement disponibles dans la plupart des laboratoires qui permettront quantification rapide et fiable de la densité des cheveux chez la souris avec des cheveux bruns ou noirs. Cette méthodologie a été testée dans des modèles murins de l&#39;alopécie induite par la chimiothérapie, la pelade, l&#39;alopécie et de Waxing. Un protocole détaillé est présenté pour l&#39;exécution de ces mesures, y compris les données de validation de C57BL / 6 et C3H / HeJ souches de souris. Comme cette technique repose sur la détection de l&#39;absorption de lumière à partir de pigments dans la tige du cheveu, il ne peut être utilisé pour détecter la croissance des cheveux chez les souris blanches ou des souris albinos.

<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

L&#39;alopécie est une forme courante de perte de cheveux qui peut se produire dans de nombreuses conditions différentes, y compris le dessin male perte de cheveux, le syndrome des ovaires polykystiques, et la pelade. L&#39;alopécie peut également se produire comme un effet secondaire de la chimiothérapie chez les patients cancéreux. Dans cette étude, notre objectif était de développer une méthode cohérente et fiable de quantifier la perte de cheveux chez la souris, ce qui permettra aux enquêteurs d&#39;évaluer et de comparer de nouvelles approches thérapeutiques pour ces différentes formes d&#39;alopécie précision. Le procédé utilise un imageur de gel standard pour obtenir des images et le processus de souris, mesurant l&#39;absorption de lumière, qui se produit en proportion approximative de la quantité de cheveux noir (ou gris) sur la souris. Les données qui ont été quantifiés de cette manière peuvent ensuite être analysés en utilisant des techniques statistiques standard (ce est à dire, analyse de la variance, T-test). Cette méthodologie a été testée dans des modèles murins de l&#39;alopécie induite par la chimiothérapie, la pelade et l&#39;alopécie de l&#39;épilation. Dans ce rapport, le protocole détaillé est presENTED pour effectuer ces mesures, y compris les données de validation de C57BL / 6 et C3H / HeJ souches de souris. Cette nouvelle technique offre un certain nombre d&#39;avantages, y compris relative simplicité d&#39;application, le recours à l&#39;équipement qui est facilement disponible dans la plupart des laboratoires de recherche, et en appliquant une évaluation objective et quantitative qui est plus robuste que les évaluations subjectives. L&#39;amélioration de la quantification de la croissance des cheveux chez la souris permettra d&#39;améliorer l&#39;étude des modèles de l&#39;alopécie et de faciliter l&#39;évaluation de nouveaux traitements prometteurs dans les études précliniques.

Cette technique a été validée à l&#39;aide souris C3H / HeJ greffées, le modèle murin de la pelade 16. Ces animaux développent la perte de cheveux mondiale qui progresse graduellement avec le temps. Cependant, la perte de cheveux survient chez des correctifs, et peut varier d&#39;une souris pour la prochaine, en introduisant une variabilité considérable et entrave les évaluations qualitatives. Ce modèle a permis de tester les corrélations entre les mesures de la densité optique à différents end...

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.

Une nouvelle technique pour l&#39;analyse quantitative de la perte de cheveux chez la souris Utilisation de l&#39;analyse des niveaux de gris

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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8.8K vues.  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.

Vidéo: 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 et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

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

Recherche

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.

Imagerie des cellules vivantes et de l&#39;analyse quantitative des cellules embryonnaires épithéliales 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 ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Une technique non invasive cheveux d&#39;échantillonnage pour obtenir l&#39;ADN de haute qualité provenant Elusive petits mammifères

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

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.

Quantitatives de fluorescence Live Cell-microscopie Analyse de la levure à 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.

Analyse quantitative des migrations au hasard des cellules en utilisant la microscopie vidéo time-lapse

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.

Un flux de travail de remise en forme Analyse quantitative

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.

Analyse rapide de la génétique des épithélio-mésenchymateuse signalisation lors de la régénération des cheveux

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.

Analyse quantitative de l&#39;autophagie en utilisant avancée Fluorescence Microscopy 3D

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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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 ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

Extraction et analyse de cortisol de l&#39;homme et de singe cheveux

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...