La ricerca descritta in questo studio è stato sostenuto da una borsa della National Science Foundation (DEB-0805818) per WAF, AWS e ID Questo studio è stato supportato anche da borse di IOS-0845847 per AWS, IOS-0919855 per AWS e ID e IOS-0920720 per WAF

<html> 1. Allevamento e la manipolazione della dieta di produrre variazioni nelle dimensioni del corpo e ali dimensioni Razionale e panoramica. L&#39;espressione dei rapporti di scala dipende in ultima analisi, sui processi di sviluppo normativo che producono variazione di organo finale e dimensioni del corpo. Questi processi sono meglio descritti dal lavoro di insetti completamente metamorfiche (cioè olometaboli) come D. melanogaster, dove gli organi crescere come indifferenziato &#39;dischi immaginali&#39; all&#39;interno del grub-come larve 11,15-17. In Drosophila, organo finale e dimensioni del corpo sono regolati da una crescita durante gli stadi larvali, larve di crescere attraverso il instar prima e la seconda larvale fino a raggiungere un peso minimo vitale per eclosion (MVW E) verso l&#39;inizio del terzo larve instar 18. La fame prima di raggiungimento di MVW E impedisce eclosion adulti. Tuttavia, le larve che sono affamati di MVW E generare adulti praticabile, anche se a una dimensione molto piccola. Questo perché riduce la nutrizione durante lo sviluppo del corpo riduce finale e le dimensioni degli organi, non solo le mosche ma in quasi tutti gli animali 19-21. Manipolando la disponibilità di cibo dopo MVW E è raggiunto, ma prima di impupamento si verifica, è possibile indurre estrema plasticità fenotipica nella dimensione corporea che supera notevolmente altre fonti di variazione (ad esempio, singole variazioni genetiche). <ol><li> Le uova sono raccolti dalla popolazione per essere fenotipizzati, suddiviso in lotti di 50 e collocati in fiale contenenti 10 ml di cibo volare standard. Perché deposizione delle uova avviene di continuo, le uova vengono raccolti ogni 24 ore per tre giorni, producendo tre coorti di età. </li><li> Quando la più antica coorte raggiunge la fase erranti o ha appena iniziato a pupate, la manipolazione dieta è applicata. A questo punto, la più antica coorte è ben oltre la E MVW, il più giovane di coorte è molto vicino alla E MVW e la coorte rimanente è a metà strada tra questi due estremi. Così, l&#39;eliminazione degli alimenti, a questo punto produrrà adulti molto grande dal più antico coorte, gli adulti molto piccolo dal più giovane di coorte e di dimensioni intermedie adulti della coorte rimanenti. Per rimuovere le larve dai flaconi, circa 5 ml di soluzione di saccarosio 40% si aggiunge a tutte le fiale. Per liberare le larve, il cibo è agitato per 15-20 minuti su un tavolo shaker insieme a una velocità intermedia. </li><li> Larve galleggianti vengono rimosse dalla soluzione di saccarosio con una multa (00) pennello, collocati in fiale contenenti un tappo di cotone bagnato e lasciato pupate. In alternativa, le larve possono essere immessi in un non-digeribile additivo alimentare come metilcellulosa 22. </li></ol> 2. Stima della dimensione del corpo Razionale e panoramica. Precedenti studi indicano che la lunghezza del torace è a meno di delega ideale per il formato generale del corpo 22. Invece, usiamo dimensioni pupa come misura della dimensione corporea. Dimensioni corporee massimo è fissato a la cessazione della alimentazione che precede impupamento. Di conseguenza, vi è una stretta correlazione tra le dimensioni pupa e il corpo formato adulto 23. <ol><li> Pupe sono disposti lato ventrale su vetrini da microscopio vetro e ripreso con una telecamera digitale collegata ad un microscopio da dissezione. Ogni immagine viene catturata, assegnato un codice univoco di identificazione e trasferito su un computer tramite un live feed di collegare la fotocamera digitale al computer. </li><li> Ripreso pupe vengono spostati a singoli epitubes 2 ml contenente 1 ml di cibo volare, etichettati con il codice unico per l&#39;individuo, e forato con prese d&#39;aria per permettere lo scambio di gas. </li><li> Dimensioni pupa è stimato come il numero di pixel della silhouette pupa. Metriche altro formato potrebbe essere utilizzato anche. </li></ol> 3. Trait (ala) dimensioni stima Razionale e panoramica. Per ali immagine, usiamo un &#39;ala grabber&#39; 24, che tira e tiene l&#39;ala di un live, perpendicolare volare anestetizzato al suo corpo. Pressato tra due pezzi di vetro, questo permette l&#39;ala di essere ripreso come un oggetto bidimensionale. <ol><li> Le mosche sono leggermente anestetizzati con CO 2 e collocati in posizione nel grabber ala di un microscopio da dissezione dotato di una fotocamera digitale che è collegato a un computer. </li><li> L&#39;ala è ripreso e salvato sotto il codice unico per l&#39;individuo. </li><li> Dimensioni ala può essere stimato utilizzando il minimo poligono convesso che circoscrive marginale punti di riferimento fissi o come il numero di pixel del profilo alare. </li></ol> 4. Scaling rapporto di stima <ol><li> I dati sono aggregati con coorti, ma separati per sesso e trasformati in logaritmo. </li><li> Rapporti di scala sono adatti ai dati utilizzando dimensioni di tipo II (cioè ridotto principale asse) di regressione (vedi recensione in 12) e dei parametri stimati per ogni gruppo. </li></ol> 5. Rappresentante dei risultati: tenda &quot;&gt; I nostri manipolazione dieta produce una vasta gamma di dimensioni del corpo (Figura 1) e dimensioni tratto (Figura 2). Tracciare i tratti accedere trasformato uno contro l&#39;altro rivela il loro modello di covariazione all&#39;interno di un dato gruppo biologico e permette di montare il rapporto di scala al gruppo (Figura 3). estrazione dei parametri del rapporto di scala, che descrivono come scale di forma con la dimensione corporea permette di confronto su come covaries forma con dimensioni tra i gruppi. <img alt="Figura 1" src="/files/ftp_upload/3162/3162fig1.jpg" /> Figura 1. Pupa dagli estremi della distribuzione delle dimensioni prodotta attraverso la manipolazione dieta dopo le persone a realizzare la E MVW. Le immagini sono reali pupa mostrato a sinistra con le loro sagome corrispondenti per la misurazione delle dimensioni pupa a destra. Barra di scala è 1 mm. <img alt="Figura 2" src="/files/ftp_upload/3162/3162fig2.jpg" /> Ali Figura 2. Tratti da estremi della distribuzione delle dimensioni del corpo prodotta attraverso la manipolazione dieta dopo le persone a realizzare la E MVW. Immagini ala reale vengono visualizzati sulla sinistra con le loro sagome corrispondenti per la misurazione delle dimensioni ala mostrato a destra. Barra di scala è 1 mm. <img alt="Figura 3" src="/files/ftp_upload/3162/3162fig3.jpg" /> Figura 3. Wing-corpo scala dimensioni per Drosophila melanogaster per sesso. I dati del log-trasformati per gli individui di ogni sesso, che sono stati completamente alimentato (occhiaie), affamati uno (cerchi grigi) o due giorni (cerchi aperti), come larve. Tipo di linea di regressione II è indicato per ciascun sesso. Tra ogni fascia di età, declina in superficie alare media di ~ 0,1 mm (~ 7%) e ~ 0.2mm (~ 8%) si verifica, aumento del numero totale dei fenotipi osservati. Le regressioni mostrano che le femmine sono leggermente hypoallometric per questo rapporto di scala (pendenza di 0,96) mentre i maschi sono leggermente hyperallometric (pendenza 1,06).

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W., Frankino, W. A., Flatt, T., Nijhout, H. F., Emlen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+shape:+The+developmental+regulation+of+static+allometry+in+insects.">Size and shape: The developmental regulation of static allometry in insects.</a> BioEssays. 29, 536-548 (2007).</li><li>Frankino, W. A., Emlen, D., Shingleton, A. W., Garland, T., Rose, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+approaches+to+studying+the+evolution+of+morphological+allometries:+The+shape+of+things+to+come.">Experimental approaches to studying the evolution of morphological allometries: The shape of things to come.</a> Experimental evolution: Concepts, methods, and applications. , (2009).</li><li>Emlen, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body-size+regulation:+Combining+genetics+and+physiology.">Body-size regulation: Combining genetics and physiology.</a> Curr. Biol. 15, 825-827 (2005).</li><li>Nijhout, H. F., Davidowitz, G., Roff, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+the+mechanism+that+controls+body+size+in+Manduca+sexta.">A quantitative analysis of the mechanism that controls body size in Manduca sexta.</a> J. Biol. 5, 16.11-16.15 (2006).</li><li>Davidowitz, G., Nijhout, H. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+regulation+of+the+insulin-like+growth+factors.">Nutritional regulation of the insulin-like growth factors.</a> Endocr. Rev. 15, 80-101 (1994).</li><li>Boots, M., Begon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resource+limitation+and+the+lethal+and+sublethal+effects+of+a+viral+pathogen+in+the+Indian+meal+moth,+Plodia+interpunctella.">Resource limitation and the lethal and sublethal effects of a viral pathogen in the Indian meal moth, Plodia interpunctella.</a> Ecol. Entomol. 19, 319-326 (1994).</li><li>Shingleton, A. W., Mirth, C. K., Bates, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+model+of+static+allometry+in+holometabolous+insects.">Developmental model of static allometry in holometabolous insects.</a> Proc. R. Soc. B. 275, 1875-1885 (2008).</li><li>Stern, D. L., Emlen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+basis+of+allometry+in+insects.">The developmental basis of allometry in insects.</a> Development. 126, 1091-1101 (1999).</li><li>Emlen, D. J., Allen, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+to+phenotype:+Physiological+control+of+trait+size+and+scaling+in+insects.">Genotype to phenotype: Physiological control of trait size and scaling in insects.</a> Integr. Comp. 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Nessun conflitto di interessi dichiarati.

Scaling morfologico è stato oggetto di intensi studi in biologia evolutiva per quasi un secolo. Più di recente, la base prossima della regolazione della crescita e scalabilità è diventato sempre più importante nello sviluppo, fisiologia e genetica (ad esempio, visualizzare le recensioni in 12,15,23-25). La nostra metodologia consente la valutazione dei rapporti di scala morfologica su tutta la gamma dei caratteri e dimensioni del corpo. Questo può essere importante, come la dimensione caratteristica potrebbe non scala linearmente con la dimensione del corpo (ad esempio, 13,14). Tuttavia, questo può non essere evidente se gli animali sono allevati solo in condizioni ideali di laboratorio. Anche se il nostro protocollo è stato sviluppato esplicitamente per poter valutare l&#39;ala-corpo scala dimensioni in Drosophila, può essere utilizzato con trait-specifica modifica per stimare il rapporto di scala per ogni caratteristica morfologica di mosche. Allo stesso modo, la manipolazione dieta può probabilmente essere usato per produrre l&#39;intera gamma di dimensioni di tratto e corpo in altri insetti completamente metamorfica; tali applicazioni richiedono semplicemente sapere quando l&#39;E MVW si ottiene applicando la manipolazione e in quel momento della ontogenesi.

experimental manipulation of body size to estimate morphological scaling relationships in drosophila

Il ridimensionamento di parti del corpo è un elemento centrale della morfologia animale 1-7. All&#39;interno delle specie, caratteristiche morfologiche devono essere correttamente proporzionata al corpo per l&#39;organismo a funzionare, gli individui più grandi hanno in genere parti del corpo più grandi e più piccoli gli individui hanno in genere parti del corpo più piccolo, in modo tale che la forma generale del corpo è mantenuto in una vasta gamma di formati corpo adulto. Il requisito di giuste proporzioni significa che gli individui all&#39;interno delle specie di solito mostrano bassa variazione di dimensione caratteristica relativa. Al contrario, la dimensione caratteristica relativa può variare enormemente tra le specie ed è un meccanismo primario attraverso il quale viene prodotta la diversità morfologica. Oltre un secolo di lavoro comparativo ha stabilito questi modelli intra-e interspecifica 3,4. Forse l&#39;approccio più utilizzato per descrivere questa variante è quello di calcolare il rapporto di scala tra le dimensioni di due caratteri morfologici mediante l&#39;equazione y = allometrica bxα, dove x e y sono le dimensioni dei due tratti, come organo e dimensioni del corpo 8 , 9. Questa equazione descrive le all&#39;interno del gruppo (ad esempio, specie, popolazione) rapporto di scala tra due tratti sia come dimensioni variabili. Log-trasformazione di questa equazione produce una semplice equazione lineare, log (y) = log (b) + αlog (x) e log-log trame della dimensione dei caratteri differenti tra individui della stessa specie tipicamente rivelare scala lineare con un&#39;intercetta di log (b) e una pendenza di α, chiamato il &#39;allometrica coefficiente di&#39; 9,10. Variazioni morfologiche tra i gruppi è descritto da differenze di intercettazioni rapporto di scala o di piste per un paio tratto dato. Di conseguenza, la variazione dei parametri dell&#39;equazione allometrica (b e α) descrive con eleganza la variazione di forma catturato nel rapporto tra organo e dimensioni del corpo all&#39;interno e tra i gruppi biologici (vedi 11,12). Non tutti i tratti di scala in modo lineare con loro o con dimensioni del corpo (ad esempio, 13,14) Quindi, i rapporti di scala morfologiche sono più informativa quando i dati sono presi dalla gamma completa dei formati tratto. Qui si descrivono come semplice manipolazione sperimentale di dieta può essere utilizzato per produrre l&#39;intera gamma di dimensioni del corpo degli insetti. Questo permette una stima del rapporto di scala completa per qualsiasi coppia di caratteri, permettendo una descrizione completa di come covaries forma con dimensioni e un confronto robusta di parametri di scalatura rapporto tra i gruppi biologici. Anche se ci concentriamo sulla Drosophila, la nostra metodologia dovrebbe essere applicabile a quasi tutti gli insetti completamente metamorfica.

Watch this Scientific Journal Video about Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila at JoVE.com

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Rapporti di scala morfologica catturare e descrivere la forma degli organismi. Vi presentiamo un metodo per misurare i rapporti di scala morfologica tutta la gamma naturale delle dimensioni del corpo degli insetti completamente metamorfica. Utilizzando un semplice manipolazione dieta aumentiamo la distribuzione delle dimensioni tratto, permettendo la descrizione accurata di come forma e dimensione co-variano.

La manipolazione sperimentale di Dimensione Corpo di Stima Relazioni scala morfologica in Drosophila</em

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body ...

experimental-manipulation-body-size-to-estimate-morphological-scaling

Research

JoVE Journal

Biology

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

13.7K Views.  University of Houston. Rapporti di scala morfologica catturare e descrivere la forma degli organismi. Vi presentiamo un metodo per misurare i rapporti di scala morfologica tutta la gamma naturale delle dimensioni del corpo degli insetti completamente metamorfica. Utilizzando un semplice manipolazione dieta aumentiamo la distribuzione delle dimensioni tratto, permettendo la descrizione accurata di come forma e dimensione co-variano.

Video: Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Experimental Approaches to Tissue Engineering

Approcci sperimentali di ingegneria dei tessuti

Ricerca

Biologia

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imaging Forma Modifica cella in Living Drosophila Embrioni

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Utilizzo di RNA-mediata strategia di alimentazione di interferenza secondo schermo per geni coinvolti nella regolazione Dimensione del corpo nel nematode<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Un metodo innovativo per Exosome quantificazione e Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Protocollo sperimentale per l'utilizzo di Drosophila come sistema modello invertebrati marini per test di tossicità in laboratorio

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Monitoraggio non invasivo della dimensione della lesione in un modello murino eterologo di endometriosi

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

Istituzione e manipolazione genetica degli organoidi epatociti murini

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

Dissezione, fissaggio e visualizzazione della Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Induzione e diversi indicatori di valutazione dell'encefalomielite autoimmune sperimentale

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...