Vorremmo ringraziare Marc Leonard presso la Carleton Immersive Media Studio per l&#39;assistenza tecnico esperto in produzione e montaggio video e il dottor Martin Bertrand presso la Carleton Immersive Media Studio / Laboratorio rigenerazione neurale per l&#39;assistenza modello visivo. Gli autori ringraziano il parere degli esperti del Dr. Marilyn Keaney e tutto il suo personale dedicato presso l&#39;Università di Ottawa e Animal Care Servizi veterinari. Questo lavoro è stato finanziato dal Canadian Institute of Health Research (CIHR, MOP 62826) per SALB, l&#39;Istituto CIHR di invecchiamento e l&#39;iniziativa di formazione strategica in Ricerca Salute / Programma di Formazione in CIHR Lipidomica neurodegenerative (TGF 96121) per SALB e SF, Canadian Foundation per l&#39;innovazione a SF, Ontario Trust Innovation a San Francisco, e Autodesk ricerca a SF. ACM riceve un CIHR Banting e Best premio di dottorato. NV riceve una borsa di studio post-professionale da Institute of Aging e Programma di Formazione in CIHR Lipidomica neurodegenerative.

1. Reagenti Preparazione <ol><li> Per sterile lavanda vaginale, autoclave acqua bidistillata (DDH 2 O) e conservare in un contenitore ermeticamente chiuso a temperatura ambiente fino al momento dell&#39;uso. </li><li> Per la valutazione citologica, aggiungere 0,1 g di polvere cristalvioletto a 100 ml di DDH 2 O. Mescolare bene. Cristalvioletto macchia (0,1%) può essere conservato in un contenitore ermeticamente sigillato a temperatura ambiente fino a quando necessario. </li></ol> 2. Raccolta di cellule vaginali (Lavage vaginale) <ol><li> Posizionare una lampadina lattice sulla fine di una sterile 200 microlitri punta e di elaborare circa 100 ml di sterile DDH 2 O utilizzando le gradazioni sulla punta come linea guida del volume. </li><li> Sollevare il mouse fuori dalla sua gabbia e metterla sulla tramoggia gabbia (coperchio) con la sua estremità posteriore / posteriore verso di voi. </li><li> Afferrare saldamente la coda e sollevare la parte posteriore. Il mouse avrà ora solo le zampe anteriori afferrare la tramoggia. A questo punto il mouse può urinare. Se è così, Attendere fino a quando si ferma minzione. In caso di urina a sinistra all&#39;ingresso del canale vaginale, si consiglia di sciacquare l&#39;apertura con eccesso di DDH 2 O con una punta separato (cioè, non la punta del prelievo del campione). </li><li> Posizionare l&#39;estremità del 2 DDH O pieno di punta all&#39;apertura del canale vaginale, avendo cura di non penetrare l&#39;orifizio vaginale come (e cervicale), la stimolazione può indurre pseudogravidanza nei ratti 1,2. Rapporti recenti suggeriscono i topi sono meno sensibili a questo effetto comunque si dovrebbe aver cura di ridurre al minimo il grado di invasività in analisi ripetute 3. </li><li> Delicatamente premere lo stantuffo per espellere un quarto alla metà del volume di acqua (~ 25-50 microlitri) in apertura del canale vaginale. Il liquido spontaneamente aspirare nel canale di inserimento senza punta. Rilasciare lentamente la pressione esercitata sul bulbo. Il fluido si ritirerà nuovamente dentro la punta. Evitare pressione rilasciando troppo velocemente per evitare l&#39;aspirazione di liquidonel bulbo. Una punta filtrata può essere utile a questo scopo. </li><li> Ripetere i tempi delle fasi precedenti 4-5 usando la punta stessa, bulbo, e fluido per ottenere un numero sufficiente di cellule in un singolo campione. </li><li> Posizionare il fluido sul vetrino, e consentire l&#39;striscio asciugare completamente a temperatura ambiente. Una volta asciutto, questi strisci estro può essere colorato immediatamente o conservate e colorate in un secondo momento. </li></ol> 3. Colorazione citologica utilizzando Crystal Violet * 4 <ol><li> Posizionare il vetrino a secco in una vaschetta Coplin (o da altro vaso colorazione simile) contenente il cristalvioletto colorare per 1 min. </li><li> Rimuovi per un barattolo secondo Coplin contenente DDH 2 O. Lavare il vetrino con DDH 2 O per 1 min. Ripeti. </li><li> Rimuovere l&#39;eccesso DDH 2 O dai bordi del vetrino con leggeri tergicristallo tessuto, evitando il contatto con lo striscio colorato. </li><li> Pipettare circa 15 ml di glicerolo in cima al coperchio e striscioscivolare. In alternativa, altri reagenti istologici di montaggio può essere utilizzata per ottenere un più permanente, non-diffondente macchia. </li></ol> * Il metodo di colorazione qui descritto è il più semplice procedura che può essere eseguita in qualsiasi laboratorio. Altri metodi in grado di fornire ulteriori dettagli. Ad esempio, utilizzando la colorazione Papanicolaou, la maturità di nucleate cellule epiteliali possono essere distinti con le cellule meno mature le cellule colorate con turchese e più maturi rosa o arancione-macchiati. Queste differenze possono essere utilizzati per organizzare proestrus presto o tardi. 4 4. Citologia Vaginale <ol><li> Esaminare lo striscio al microscopio ottico per determinare i tipi di cellule presenti. Esame microscopico dovrebbe essere fatto immediatamente dopo colorazione come cristalvioletto diffonderà dalle cellule nel tempo quando si utilizza glicerolo per coprioggetto. Microfotografie deve essere assunto al momento di analisi per documentare citologia. </li><li> Iniziamo ad esaminare la entirstriscio e con un ingrandimento inferiore. Selezionare un&#39;area rappresentativa e passare a un ingrandimento maggiore. Vedrete cornified squamose cellule epiteliali, leucociti, e / o nucleate cellule epiteliali (risultati rappresentativi, Figura 1A-C). Il rapporto tra presente cellule vi permetterà di determinare la fase estrale del mouse al momento del prelievo del campione (risultati rappresentativi, figura 1D-G) e il suo stato ormonale immediata (discussione, Figura 2). </li></ol> 5. Risultati rappresentativi Citologia: Tre tipi cellulari primarie può essere rilevato in campioni di striscio vaginale: (1) nucleate cellule epiteliali (Figura 1A), (2) cornified cellule epiteliali squamose (Figura 1B), e (3) leucociti (Figura 1C). Nucleate cellule epiteliali hanno un citoplasma poco sporca, più scuro macchiato membrana plasmatica, e un nucleo ovale ( <strong&gt; Figura 1A). Cornified cellule epiteliali squamose sono uniformemente macchiato, più poligonale loro predecessori nucleate epiteliali, e mancanza di un nucleo (Figura 1B). Leucociti polimorfonucleati può essere distinto da cellule epiteliali dalla loro forma irregolare, scuro macchiato nuclei polimorfi, e le piccole dimensioni (Figura 1C, frecce nere). Contaminazione dovrebbe essere presente nelle urine striscio, cristalli di acido urico sono prontamente rilevato dalle loro strutture cristalline dissimili a qualsiasi tipo di cella attesi (Figura 3). In questo caso, e la rilevazione oscura di tipo cellulare predominante, lo striscio deve essere eliminato e non utilizzati a scopo di stadiazione. Staging: Il rapporto relativo di tipi di cellule osservate negli strisci può essere utilizzato per identificare la fase del ciclo estrale del mouse, il giorno della raccolta del campione (Figura 1D-G). Durante proestro, le cellule sono quasi esclusivamentegruppi di rotondo, ben formato nucleate cellule epiteliali (Figura 1D, cellula rappresentante indicato dalla freccia bianca). Durante l&#39;estro, le cellule sono prevalentemente cornified cellule epiteliali squamose, presenti in gruppi densamente (Figura 1E, cellula rappresentante indicato dalla freccia). Durante metestrus, piccoli leucociti scuro macchiato predominano (Figura 1F, cella rappresentante indicato dalla freccia nera). Corneo cellule epiteliali squamose possono essere osservati, spesso in frammenti, (Figura 1F, cellula rappresentante indicato dalla freccia nera). Durante il diestro, cornified rare cellule epiteliali squamose possono essere ancora presenti (Figura 1G, cella rappresentante indicato dalla freccia nera), ma ancora predominano leucociti (Figura 1G, cella rappresentante indicato dalla freccia nera). Metestrus può essere distinto da diestro dalla comparsa di cellule nucleate epiteliali in diestro ( <stron<html> g&gt; Figura 1G, cellula rappresentante indicato dalla freccia bianca). <img src="/files/ftp_upload/4389/4389fig1.jpg" alt="Figura 1" /> Figura 1. Valutazione citologica di strisci vaginali può essere utilizzato per identificare fase estrale tre tipi principali di cellule vengono rilevati in campioni di striscio vaginale:. (A) nucleate cellule epiteliali, (B) cornified cellule epiteliali squamose, e (C) leucociti. Il rapporto di questi tipi di cellule presenti nel striscio può essere utilizzato per identificare in topi (D) proestrus, (E) estro, (F) metestrus, o (G) come descritto in diestro risultati rappresentativi. Punte di freccia nere in E, F e del punto G alla rappresentanza cornified cellule epiteliali squamose. Frecce nere in C, F e del punto G alla leykocytes rappresentativi. Frecce bianche in D e rappresentante evidenziare G nucleate cellule epiteliali. . &quot;Alt =&quot; jpg Figura 2 &quot;/&gt; Figura 2. Citologia striscio vaginale riflette eventi endocrini sottostanti. Dettagli sono forniti anche in discussione. <a href="http://www.jove.com/files/ftp_upload/4389/4389fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img src="/files/ftp_upload/4389/4389fig3.jpg" alt="Figura 3" /> Figura 3. Cristalli di acido urico possono essere presenti di colorazione viola cristallo di urina-campioni contaminati. (A) I cristalli sono trasparenti e possono essere di varie dimensioni (frecce e regione scatola ingrandite in (B)). Nessun cellule sono presenti in questo campo. Dovrebbe contaminazione cristallo acido urico entro campi usati per la colorazione citologica essere presente, può essere difficile identificare con precisione tipi di cellule presenti e lo striscio deve essere scartato. Barre di scala = 50 micron.

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Gli autori dichiarano assenza di conflitto di interessi. Tutti gli esperimenti sugli animali sono stati eseguiti in stretta conformità con le linee guida e regolamenti stabiliti dalla University of Ottawa Animal Care Comitato e il Canadian Council on Animal Care.

Questi cambiamenti nella tipologia cellule sono indicativi di eventi endocrini sottostanti. La fase proestro del ciclo estrale corrisponde alla fase follicolare umano del ciclo mestruale e 5 è definito da un pre-ovulatoria aumento circolanti 17-β-estradiolo livelli 6, nonché un aumento piccolo prolattina 7 (figura 2, proestro, pannello di sinistra). L&#39;incremento 17-β-estradiolo stimola indirettamente gonadotropina-releasing ormone neuroni dell&#39;ipotalamo e setto che, a loro volta, attivano cellule responsive nella pituitaria anteriore per rilasciare l&#39;ormone luteinizzante e ormone follicolo-stimolante nella circolazione 8,9 (figura 2 , proestro, pannello di sinistra). In strisci vaginali prelevati da animali in proestro, le cellule sono cellule nucleate quasi esclusivamente ovali epiteliali (Figura 1D, Figura 2, proestro, pannello di destra). Il picco nel follicolo-stimolante HORMuno dei livelli dei segnali l&#39;ovulazione e l&#39;entrata in estro 10,11. Durante l&#39;estro, 17-β-estradiolo livelli di livelli di prolattina e declino di picco 6,7 (Figura 2, Estrus, pannello di sinistra). Strisci vaginali sono caratterizzati da rilevamento quasi esclusivo di forma irregolare cornified cellule epiteliali squamose spesso in ciuffi (Figura 1E, Figura 2, Estrus, pannello di destra). Entrata in metestrus coincide con un continuo aumento dei livelli dell&#39;ormone progesterone 6 e corrisponde all&#39;inizio della fase luteale umano 12 (Figura 2, Metestrus, pannello di sinistra). Come i livelli di progesterone inizia a salire e c&#39;è un piccolo aumento in 17-β-estradiolo livelli in risposta all&#39;attivazione del corpo luteo 6,13,14 (Figura 2, Metestrus, pannello di sinistra). I tipi di cellule presenti in strisci vaginali durante questa fase sono frammentate, le cellule epiteliali e leucociti cornified più scure più piccoli colorati (Figure 1F, Figura 2, Metestrus, pannello di destra). Infine, entrata in diestro in topi verifica e livelli circolanti progesterone picco 6, corrispondente alla fase tardiva umana luteale 12. Regressione del corpo luteo porta ad una conseguente diminuzione forte livelli di progesterone 15,16 (Figura 2, diestro, pannello di sinistra). Leucociti predominano negli strisci durante diestro. La frequenza di cornified cellule epiteliali è ridotto e nucleate cellule epiteliali iniziano a essere rilevato appena prima transizione proestrus (Figura 1G, Figura 2, diestro, pannello di destra). In sintesi, questo semplice protocollo di routine può essere utilizzato per stimare quotidiane fluttuazioni ormonali e stabilire fase estrale nei topi sperimentali, senza alterare lo stato riproduttivo se le seguenti precauzioni vengono prese. Il campionamento deve essere effettuato non più di una volta al giorno utilizzando il protocollo non invasivo described qui rispetto alla penetrazione ripetuta del canale vaginale, l&#39;aspirazione, e agitazione. Questo può causare irritazione vaginale, un 17 risposta infiammatoria conseguente leucociti e altri tipi di cellule di essere presente in strisci che possono confondere valutazione citologica. Inoltre, anche in colonie alloggiati femmine, è normale vedere diestro estesi e le fasi di estro nei topi diversi, come pure l&#39;induzione di anestrous 18 e questa identificazione è utile per l &#39;interpretazione di impatto ormonale riproduttiva, il sesso e gli studi sulle malattie. La variabilità della durata del ciclo viene introdotto anche con l&#39;età e con le differenze degli alloggi nelle colonie (individuale o di gruppo-housing) di femmine 7,19-21. Le femmine alloggiati nelle donne solo colonie può cessare in bicicletta e in uno stato di prolungato diestro 18,22,23, anche se il ciclismo può essere ripristinato in caso di esposizione a gabbie pretrattati con urina maschile per suscitare ciclismo 24,25. Pertanto, per stabilire individuadurata del ciclo l per un mouse dato, si raccomanda che le valutazioni non invasive descritte qui effettuata giornalmente, con cura, fino a due cicli completi si osservano.

<table border="1"><tbody><tr><td> Nome </td><td> Azienda </td><td> Catalogo # </td></tr><tr><td> Sterili 200 puntali pl </td><td> DiaMed </td><td> E340901 </td></tr><tr><td> Lattice lampadina (1 ml) </td><td> Pescatore </td><td> 03-488-21 </td></tr><tr><td> Vetro microscopio diapositive </td><td> Pescatore </td><td> 12-550-15 </td></tr><tr><td> Crystal Violet macchia (25 g) </td><td> Pescatore </td><td> C581-25 </td></tr><tr><td> Leggeri tessuti Panni </td><td> VWR </td><td> 82003-820 </td></tr><tr><td> Glicerina </td><td> Pescatore </td><td> BP229-1 </td></tr><tr><td> Microscopio di vetro di copertura (22x30) </td><td> Pescatore </td><td> 12-544A </td></tr></tbody></table>

performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification

Un mezzo rapido di valutare lo stato riproduttivo nei roditori è utile non solo nello studio della disfunzione riproduttiva, ma è necessario anche per la produzione di nuovi modelli murini di malattia e indagini nella regolazione ormonale della degenerazione del tessuto (o rigenerazione) dopo sfida patologica. Il murino riproduttiva (o estro) ciclo è diviso in 4 fasi: proestro, estro, metestrus e diestro. Fluttuazioni definiti dei livelli circolanti degli steroidi ovarici 17-β-estradiolo e progesterone, le gonadotropine luteinizzante e follicolo ormoni stimolanti, e la transizione di ormone prolattina luteotropic segnale attraverso queste fasi riproduttive. Variazioni tipologia delle cellule all&#39;interno del canale vaginale murino riflettere questi eventi endocrini sottostanti. Valutazione giornaliera del rapporto relativo nucleate cellule epiteliali, cellule epiteliali squamose cornified, e leucociti presenti negli strisci vaginali può essere utilizzato per identificare estrale murinefasi. Il grado di invasività, però, impiegato nella raccolta di questi campioni possono alterare lo stato riproduttivo e provocare una risposta infiammatoria che può confondere la valutazione citologica di strisci. Qui, descriviamo un semplice, non invasivo protocollo che può essere utilizzato per determinare la fase del ciclo estrale di un topo femmina senza alterare il suo ciclo riproduttivo. Ci dettaglio come distinguere tra le quattro fasi del ciclo estrale di raccolta e l&#39;analisi della tipologia cellula predominante negli strisci vaginali e si mostra come questi cambiamenti possono essere interpretati in relazione allo stato endocrino.

Watch this Scientific Journal Video about Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification at JoVE.com

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

Qui, descriviamo come identificare lo stadio della murino riproduttiva (proestro, estro, metestrus o diestro) per semplice, non invasivo raccolta e la valutazione citologica di campioni di striscio vaginale. Abbiamo inoltre descritto come citologia vaginale riflette livelli circolanti ormonali alla base di transizione attraverso il ciclo riproduttivo murino.

Esecuzione Lavage vaginale, di colorazione viola di cristallo, e la valutazione citologica vaginale per l&#39;identificazione ciclo estrale Staging mouse

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

performing-vaginal-lavage-crystal-violet-staining-vaginal-cytological

Research

JoVE Journal

Biology

110.1K Views.  Neural Regeneration Laboratory and Ottawa Institute of Systems Biology. Qui, descriviamo come identificare lo stadio della murino riproduttiva (proestro, estro, metestrus o diestro) per semplice, non invasivo raccolta e la valutazione citologica di campioni di striscio vaginale. Abbiamo inoltre descritto come citologia vaginale riflette livelli circolanti ormonali alla base di transizione attraverso il ciclo riproduttivo murino.

Video: Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Un reversibile, metodo non invasivo per le misure di resistenza delle vie aeree e liquido di lavaggio broncoalveolare di campionamento nei topi

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Ricerca

Biologia

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Valutazione dello Sviluppo ghiandola mammaria e funzione in modelli murini

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

Analisi del ciclo cellulare dal vivo di<em&gt; Drosophila</em&gt; I tessuti utilizzando l&#39;Sintonizzatevi Acoustic Concentrandosi Cytometer e Vybrant DyeCycle Violet DNA Stain

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing per identificazione microbica e caratterizzazione

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

L&#39;analisi citologica della spermatogenesi: Preparati Live e fisse di<em&gt; Drosophila</em&gt; Testicoli

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

Metodi per la gestione temporanea Pupal periodi e misura di ala pigmentazione della Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-inner Cell Mass iniezione delle cellule staminali embrionali porta a tassi più elevati di chimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Quantificazione dell'abbondanza del lipido e valutazione della distribuzione del lipido in Caenorhabditis elegans Nilo rosso e macchie di olio rosso O

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Identificazione e dissezione dei diversi depositi di adiposi del mouse

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...