Ringraziamo Haruya Ohno, Kosaku Shinoda, Louis Sharp, Emi Tomoda, e Lauren Ruiz per la discussione, assistenza tecnica e assistenza editoriale sul manoscritto. Questo lavoro è stato supportato anche da finanziamenti del NIH (DK087853), dal Programma per la Ricerca Biomedica e dal Breakthrough Asubio Pharm Inc. per SKULA stato sostenuto da una SHARE borse di dottorato di ricerca presso l&#39;Università di Copenhagen e l&#39;Unione europea del 7 ° PQ progetto Diabat (HEALTH-F2 -2.011-278373) di Lise Madsen e Karsten Kristiansen. Riconosciamo inoltre il centro DERC sovvenzione (NIH DK063720 P30).

1. Preparare Medio Digestione Fare 5 ml per 5 topi per tessuti (circa 1 ml / 1 tessuto adiposo g). <ol><li> Pesare in enzimi digestivi: - Collaginase D: 1,5 u / ml (1108874103, 1 g, Roche, 70334223) - Dispasi II: 2.4 U / ml (04942078001, 0980 mg / lyo, Roche, 11466200) </li><li> Aggiungere 25 ml di PBS e mescolare bene per sciogliere </li><li> Aggiungere CaCl 2 appena prima digestione del tessuto ad una concentrazione finale di 10 mM </li></ol> 2. Dissect tessuto adiposo di topi <ol><li> Dissezione deve essere eseguito con cura, e subito dopo i topi (6-8 settimane) vengono sacrificati. Lavora con etanolo sterile spruzzato sulla pelliccia del mouse prima di aprirlo. Posizionare il tessuto direttamente in PBS pulito, separato BAT interscapolare e sottocutaneo inguinale WAT. </li><li> Per BAT interscapolare: hanno il mouse posizionato con il dorso affrontato, sezionato lungo la schiena e fino al collo. Il marronetessuto adiposo si trova proprio sotto la pelle tra le spalle (interscapolare). BAT può essere visto come due lobi, a forma di farfalla. Un sottile strato di grasso bianco che copre il BAT dovrebbe essere accuratamente rimosso. </li><li> Per via sottocutanea WAT ​​(inguinale WAT): rimuovere la pelle. Inguinale WAT è situato direttamente sotto la pelle su entrambi i lati, a partire quasi sulla schiena e costeggiando, all&#39;interno delle cosce e in basso, verso il testicolo. </li></ol> 3. Tagliare e Digest tessuto adiposo <ol><li> Rimuovere rapidamente ogni contaminazione, come i capelli, muscolo scheletrico e tessuto connettivo dalle sezioni di tessuto. </li><li> Asciugare su carta velina in fretta per non diluire media dissezione con PBS e metterlo su un piatto asciutto. </li><li> Aggiungere mezzo digestione (aggiunto CaCl 2 prima della digestione) e tritare il tessuto in piccoli pezzi. Mescolare bene pipettando su e giù con una pipetta da 5 ml. Trasferire i tessuti tritati in provette da 50 ml con il resto del m digestioneedio, mescolare ancora di più pipettando su e giù. </li><li> Digest in 37 ° C con agitazione costante a 150 rpm per 40-50 min. Controllare ogni 10-15 minuti per assicurarsi che la digestione sta andando bene e per evitare sovra-digestione. </li></ol> Nota: media la digestione e l&#39;ora corrette sono importanti. Il tessuto deve essere ben digerita, ma digestione eccesso può danneggiare le cellule. 4. Sospensione cellulare Filtro <ol><li> Arrestare digestione aggiungendo 5 ml mezzo completo (DMEM/F12 FPS contenenti 10% e P / S). Le cellule dovrebbero essere quasi del tutto omogeneo. Mescolare bene pipettando. </li><li> Centrifugare a 700 xg per 10 min. </li><li> SVF ora può essere visto come una pastiglia brunastro sul fondo della provetta. Aspirare il adipociti maturi strato oleoso sulla parte superiore e maggior parte dello strato liquido - mantenere il pellet e farla sciogliere in 10 ml di mezzo completo. Mescolare bene. </li><li> Mettere un filtro cella (50-70 micron di diametro) in un nuovo 50<html> ml tubo e filtrare la sospensione cellulare. </li></ol> 5. Piatto Celle <ol><li> Trasferire sospensione cellulare in una provetta da 15 ml e centrifugare a 700 xg per 10 min. </li><li> Aspirare il terreno e risospendere pellet in 10 ml di terreno completo. Pipetta e mescolare bene. </li><li> Stimare il numero di piatti necessari. Questo può variare e dipende dalla dimensione pellet. Una regola generale è 2 di 10 piatti cm da 5 topi per inguinale WAT e uno da 10 cm per piatto BAT. </li><li> Celle a piastre sui piatti rivestiti di collagene (R &amp; S) in mezzo completo </li><li> 1-2 ore dopo la placcatura le cellule, medie aspirato, lavare due volte con PBS e aggiungere mezzo fresco. Questo passaggio è importante in quanto questo può rimuovere i globuli rossi, le cellule del sistema immunitario e altri contaminanti. </li></ol> 6. Differenziare le cellule <ol><li> Fare manutenzione e mezzi induzione. </li></ol> Manutenzione medie Completo di supporto con l&#39;aggiunta di: ent &quot;&gt; Insulin, conc finale. 5 ug / ml (5 mg / ml magazzino, *** 100 ml di acido acetico in 10 ml di H 2 O per preparare pH acqua acidificata 2,5. dissolvono insulina in acqua acidificata. Conservare a magazzino -20 ° C) 3,3 &#39;,5-triiodo-L-tironina (T3), conc finale. 1 nM (stock 10 mM, *** sciogliere T3 in 1N NaOH e aggiungere mezzo per rendere stock 10 mM. Sigma cat # T-2877) Conservare a 4 ° C, buona per una settimana Induzione media Manutenzione mezzo contenente i seguenti composti: Indometacina, conc finale. 125 micron (0,125 magazzino M in etanolo, cat Sigma # I-7378). Indometacina deve essere riscaldata a 60 ° C da sciogliere. Desametasone, conc finale. 2 ug / ml (2 mg / ml in etanolo calzino, Sigma cat # D-1756) 3-isobutil-1-metilxantina (IBMX), conc finale. 0.5 mM (0,25 magazzino M in DMSO, Sigma cat # I-5879) Rosiglitazone, finale conc. 0,5 micron(Stock 10 mM in DMSO, Sigma cat # R-2408) Nota: Assicurarsi di induzione fresco medio di volta in volta. <ol start="2"><li> Crescere adipociti primari al 95-97% confluenza in mezzo completo (confluenti ma non troppo imballato) </li><li> Cambiare regolarmente terreno completo con induzione a media (giorno 0) </li><li> Dopo 48 ore (giorno 2), cambiare medio e medio di mantenimento con rosiglitazone (0,5 micron) </li><li> Dopo ulteriori 48 ore (giorno 4), cambiare terreno di mantenimento fresco con rosiglitazone (1 mM) per altri 2-3 giorni. 6-7 giorni dopo l&#39;aggiunta del mezzo di induzione, le cellule sono completamente differenziate a maturare cellule di grasso e sono riempite con goccioline di olio. Goccioline apparirà intorno 3-4 giorni dopo l&#39;induzione. </li></ol> Nota: medio cambio ogni 2-3 giorni fino a quando le cellule sono completamente differenziate. Le cellule possono essere raccolte e l&#39;espressione di mRNA di Ucp1 e Other adipocita bruno-specifici geni può essere misurata mediante RT-PCR. Western blot sarà utilizzato per rilevare le proteine.

<ol>
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Gli autori dichiarano di non avere conflitto di interessi finanziari.

Qui vi presentiamo un sistema affidabile cellulare per studiare lo sviluppo di cellule beige / brite in colture primarie adipociti nei topi. Rispetto a diverse linee cellulari immortalizzate disponibili, questo sistema è suscettibile di offrire maggiore rilevanza alla doratura di grasso bianco in vivo. Anche se lo studio di questi adipociti primari offre alcuni vantaggi, esistono anche alcune limitazioni e preoccupazioni che sono importanti da considerare. Primo, questo sistema è ...

<html> L&#39;obesità è in aumento in tutto il mondo in modo drammatico ed è ora considerato una delle preoccupazioni più gravi per la salute pubblica 1. Questa condizione si riferisce ad un squilibrio in relazione al consumo di energia spesa e risultati in eccesso di energia memorizzata come lipidi nel tessuto adiposo bianco (WAT). WAT allargata è associata con un aumento di massa corporea e il peso, mentre il tessuto adiposo bruno ha la capacità di dissipare l&#39;energia in eccesso per produrre calore. Quindi BAT può funzionare sia come protezione contro freddo e l&#39;obesità 2,3. Questo si ottiene disaccoppiamento del trasporto di elettroni nei mitocondri di disaccoppiamento della proteina 1 (UCP1). Questa proteina è considerato un marchio di garanzia per nonshivering termogenesi nel BAT 3. Diversi studi negli ultimi anni hanno rivelato che gli esseri umani adulti hanno 4-8 BAT funzionale e, di conseguenza, la manipolazione delle BAT negli esseri umani può essere un possibile intervento terapeutico nella lotta contro l&#39;obesità e le malattie correlate. jove_content &quot;prove&gt; corrente indica che due tipi di adipociti bruni esistono in roditori,&quot; classico &quot;o&quot; pre-esistente &quot;grasso bruno si sviluppa durante la fase prenatale e forma dedicati depositi adiposi marroni in regione interscapolare e di altri tessuti periferici D&#39;altra parte. , una forma &quot;inducibile&quot; di grasso bruno (così chiamate cellule brite o beige) si sviluppa durante la fase post-natale e appare intervallati nel tessuto adiposo bianco. I due tipi di adipociti bruni sono separati da diverse origini dello sviluppo. Mentre il pre-esistente adipociti bruni derivano da myoblastsic-come Myf5 precursori, i brite / beige cellule inducibili intervallati in WAT nascono da una non-Myf5 lignaggio 9,10. Inoltre, percorsi di regolamentazione di questo tipo di cellule è probabile che sia diverso da quello Myf5 derivato marrone adipociti 11. Lo sviluppo delle cellule beige (cioè &quot;doratura&quot; di grasso bianco) può essere attivato in risposta a esposizione cronica a freddo eβ3-adrenergici agonisti o agonisti PPARy negli adulti 12-14. Le cellule beige / brite sono suscettibili di essere un bersaglio terapeutico promettente per la manipolazione di bilancio energetico complessivo e può potenzialmente diventare parte del trattamento dell&#39;obesità, di conseguenza, è importante capire precisi meccanismi molecolari e vie di segnalazione da stimoli ambientali che controllano lo sviluppo del beige cellule. Per comprendere il controllo molecolare della doratura di grasso bianco, esperimenti in vitro sono più adatti come differenziazione dei preadipociti avviene piuttosto asincrono ed è difficile rilevare le cellule in situ 15. Sebbene studi sullo sviluppo adipociti sono state finora eseguite principalmente su linee cellulari quali 3T3-L1, 3T3-F442A o HIB1, queste linee cellulari sembrano non firma molecolare delle cellule beige. D&#39;altra parte, adipociti primari isolati da sottocutaneo WAT hanno più probabilità di ricapitolare i process di doratura di grasso bianco in modo cellula autonoma. Qui forniamo un protocollo per un efficace isolamento della frazione stromale-vascolare da tessuti adiposi e per indurre l&#39;imbrunimento del grasso bianco in risposta ad agonisti PPARy. Rosiglitazone ha dimostrato di essere un mediatore particolarmente efficace di doratura in queste cellule. Come suggerito in precedenza 16, questo sistema cellulare può essere utilizzato per servire un sistema cellulare affidabile per studiare lo sviluppo delle cellule beige / brite.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagente </td></tr><tr><td> Collaginase D </td><td> Roche </td><td> 11088874103 </td><td></td></tr><tr><td> Dispasi II </td><td> Roche </td><td> 04942078001 </td><td></td></tr><tr><td> CaCl 2 </td><td></td><td></td><td></td></tr><tr><td> DMEM media </td><td> Pescatore </td><td> 10017-CV </td><td> Con 2,5 g / l di glucosio e L-glutammina senza piruvato di sodio </td></tr><tr><td> Insulina </td><td></td><td></td><td></td></tr><tr><td> T3 (3,3 &#39;,5-triiodo-L-tironina) </td><td> Sigma </td><td> T-2877 </td><td></td></tr><tr><td> Indometacina </td><td> Sigma </td><td> I-7378 </td><td></td></tr><tr><td> Desametasone </td><td> Sigma </td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td> Sigma </td><td> I-5879 </td><td></td></tr><tr><td> Rosiglitazone </td><td> Sigma </td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> Attrezzatura </td></tr><tr><td> Piatti collagene rivestite </td><td> BD </td><td> 354450 </td><td> 10 centimetri piastre </td></tr><tr><td> 70 pm filtro </td><td> BD Falcon </td><td> 352350 </td><td> Cella filtro, 70 micron nylon 1/ea </td></tr></tbody></table>

isolation and differentiation of stromal vascular cells to beige/brite cells

Adipociti bruni hanno la capacità di disaccoppiare la catena respiratoria nei mitocondri e dissipare l&#39;energia chimica sotto forma di calore. Sviluppo di adipociti bruni UCP1-positive nei tessuti adiposi bianchi (cosiddetta cellule beige o brite) è altamente indotta da una varietà di stimoli ambientali come esposizione cronica freddo o da agonisti PPARy, quindi, questo tipo di cella ha un potenziale come un obiettivo terapeutico trattamento dell&#39;obesità. Sebbene la maggior parte delle linee degli adipociti immortalizzate non può ricapitolare il processo di &quot;doratura&quot; di grasso bianco nella cultura, adipociti primari isolati da stromale frazione vascolare nel tessuto sottocutaneo adiposo bianco (WAT) forniscono un sistema affidabile cellulare per studiare il controllo molecolare di beige / brite lo sviluppo delle cellule . Qui si descrive un protocollo per l&#39;isolamento efficace preadipociti primarie e per indurre la differenziazione di cellule beige / brite in coltura. L&#39;effetto di doratura può essere valutata mediante l&#39;espressione di grasso bruno-selettivo marchioERS come UCP1.

Browning di adipociti primari può accedere misurando l&#39;espressione di mRNA di Ucp1 e altri geni grasso bruno-specifici o selettivi per RT-PCR. Presentato in figura 1 è dati di espressione genica in inguinali WAT-derivati ​​adipociti primari. Le cellule sono state indotte a differenziarsi in presenza di due diverse dosi di rosiglitazone a 50 nm e 500 nm, rispettivamente. Un sottoinsieme di cellule è stato trattato con forskolin a 10 pM per 4 ore prima della raccolta. Ciò provocherà-...

Watch this Scientific Journal Video about Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells at JoVE.com

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Primarie preadipociti bianchi isolati dal tessuto adiposo bianco nei topi possono essere differenziate in cellule del beige / brite. Presentato qui è un sistema affidabile modello cellulare per studiare la regolazione molecolare della &quot;doratura&quot; di grasso bianco.

Isolamento e la differenziazione di cellule stromali vascolari alle celle Beige / Brite

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

isolation-differentiation-stromal-vascular-cells-to-beigebrite

Research

JoVE Journal

Biology

38.4K Views.  University of California, San Francisco. Primarie preadipociti bianchi isolati dal tessuto adiposo bianco nei topi possono essere differenziate in cellule del beige / brite. Presentato qui è un sistema affidabile modello cellulare per studiare la regolazione molecolare della "doratura" di grasso bianco.

Video: Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolamento e di espansione degli animali siero gratuito di cordone ombelicale umano derivato cellule stromali mesenchimali (MSC) e cellule endoteliali progenitrici formanti colonia (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Ricerca

Biologia

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e colture primarie di cellule di topo aortica endoteliali

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolamento di CD146<sup&gt; +</sup&gt; cellule residenti del polmone mesenchimali stromali da Rat Polmoni

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

L&#39;isolamento di Murine coronarica vascolari cellule muscolari lisce

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolamento, caratterizzazione e differenziamento di cellule staminali cardiache dal cuore di topo adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Isolamento, proliferazione e differenziamento di cellule staminali derivate dall'adiposio del macaco rhesus

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolamento di miociti atriali e ventricolari murini di alta qualità per misurazioni simultanee di transitori Ca2+ e corrente di calcio di tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...