Questa ricerca è stata sostenuta in parte dal programma di ricerca intramurale del National Cancer Institute presso il National Institutes of Health, e NCI concedere UA5CA152907.

1 Raccolta di mezzi condizionati per testare Angiogenic Potenziale <ol><li> Crescere cellule primarie o immortalizzate da testare per il potenziale angiogenico o anti-angiogenica nei media siero nativi o basse e raccogliere i media condizionata. <ol><li> In alternativa, l&#39;uso condizionato mezzi immediatamente, o aliquotato e conservato a -80 ° C per diversi mesi. Utilizzare nonconditioned supporti siero nativo o partire da un controllo negativo, e usare i media non condizionata completa crescita (10% FBS, o la concentrazione del caso) come controllo positivo. In alternativa, per testare potenziali stimolatori dell&#39;angiogenesi, somministrare carbone mezzi spogliato mancano fattori di crescita con concentrazioni note di specifiche proteine ​​di interesse e di confrontare i fattori di crescita media liberi da soli. </li></ol></li></ol> 2 Preparazione di cellule endoteliali prima del dosaggio <ol><li> Due giorni prima del dosaggio, il passaggio 3B-11 celle in un nuovo fiasco T-75. Passaggio numero di cellule è importante. Questo test funziona meglio quando le cellule sono tra il secondo e il sesto passaggi. Il test non funziona bene se si usano cellule endoteliali prima del secondo o dopo il decimo passaggio. </li><li> Crescere le cellule per 24 ore in DMEM supplementato con 10% FBS, 2 mM L-glutammina, 1 mM di piruvato di sodio, 100 U / ml di penicillina e 100 mg / ml di streptomicina. </li><li> Un giorno prima del test, il siero di fame 3B-11 celle come segue: <ol><li> Aspirare il terreno dalle cellule 3B-11. </li><li> Aggiungi mezzi siero ridotto di DMEM integrato con 0,2% FBS, 2 mM L-glutammina, piruvato di sodio 1mM, 100 U / ml di penicillina, e 100 mg / ml di streptomicina. </li><li> Crescere le cellule per altre 24 ore. </li></ol></li></ol> 3 Preparazione dei reagenti Prima di Assay NOTA: le concentrazioni di BME sono altamente variabile dipendente su lotto. BME non funziona bene se la concentrazione è inferiore a 10 mg / ml, pertanto il costruttore BME deve essere contattato prima dell&#39;acquisto per garantireacquisizione di un lotto adeguatamente concentrato. <ol><li> Sbrinamento ridotto fattore di crescita BME in frigorifero. BME solidifica quando caldo, quindi deve essere mantenuto freddo prima dell&#39;uso. BME può essere conservato in frigorifero per un paio di settimane, o aliquotato e congelato a -80 ° C. </li><li> Un fattore di crescita ridotta BME è preferibile rispetto tradizionale BME. La mancanza di fattori angiogenici nel BME sarà più facile osservare l&#39;effetto che i fattori dai media condizionati hanno sulla formazione del tubo. </li><li> Scongelare supporti da testare. </li></ol> 4 Preparazione di cellule endoteliali immediatamente prima del saggio <ol><li> Lavare 3B-11 cellule in DPBS. <ol><li> Se la visualizzazione con fluorescenza o confocale microscopia è desiderato, prima di lavare 3B-11 celle, aggiungere calceina AM alle cellule endoteliali nei media ad una concentrazione finale di 2 mg / ml. Inserire cellule marcate calceina al buio a 37 ° C e 5% di CO 2 prima dell&#39;inizio del test. Sciogliere magazzino calceinaAM in DMSO e una volta calceina è diluito nei media concentrazione finale di DMSO non deve superare lo 0,1%. Dopo 30-45 min lavaggio calceina AM dalle cellule (punto 4.1) e continuare con l&#39;esperimento segue. </li></ol></li><li> Trypsinize cellule con l&#39;aggiunta di 1 ml di tripsina-EDTA per T-75 pallone. </li><li> Dopo tripsinizzazione è completa, neutralizzare tripsina da risospendere le cellule in un volume finale di 10 ml di DMEM, 10% FBS, 2 mM L-glutammina, piruvato di sodio 1 mM, 100 U / ml di penicillina, e 100 mg / ml di streptomicina. </li><li> Celle filtranti attraverso 100 micron filtro cella per rimuovere grumi. </li><li> Contare le cellule e risospendere le cellule ad una concentrazione finale di 7,5 x 10 6 cellule / ml di DMEM, 10% FBS, 2 mM L-glutammina, 1 mM di piruvato di sodio, 100 U / ml di penicillina e 100 mg / ml di streptomicina. </li></ol> 5 Tubo saggio Formazione <ol><li> Mettere 24 pozzetti e 1 ml punte in frigorifero o su ghiaccio per 20-30 minuti prima di caricare la piastra in modo che la BME non PREMaturely solidificarsi. </li><li> Dispensare 250 ml di fattore di crescita ridotto BME in ciascun pozzetto di una piastra da 24 pozzetti. Evitare di creare bolle non premendo la pipetta e arrivare al capolinea, mentre l&#39;erogazione. Fare attenzione che sufficiente BME rimane al centro del pozzo quando le forme menisco; se la matrice è troppo sottile, le cellule si formano solo un monostrato. </li><li> Incubare la piastra a 24 pozzetti a 37 ° C e 5% di CO 2 per 30 min a solidificare BME. </li><li> Una volta BME ha fissato, mescolare circa 300 ml di mezzi condizionati con 10 ml di risospese 3B-11 cellule (circa 75.000 celle). <ol><li> Regolare il volume dei media condizionati e il numero di cellule piastrate a seconda del tipo di cellule endoteliali utilizzato, e testare le diluizioni seriali dei media condizionati per dimostrare l&#39;attività. Se le cellule confrontando coltivate in condizioni diverse, normalizzare il volume mezzi condizionati al numero di celle che sono stati utilizzati per condizionare i media. </li></ol></li><li> Tubi will cominciano a formarsi entro 2-4 ore. A seconda della concentrazione di fattore angiogenico nei media condizionata, formazione del tubo picco potrebbe verificarsi tra 3 e 12 ore e la tempistica del test dovrà essere ottimizzato. Se si lavora con le cellule endoteliali primarie invece di cellule immortalizzate, formazione del tubo iniziale può essere ritardato di diverse ore. <ol><li> Tubi spesso iniziano a deteriorarsi entro 18 hr e cellule endoteliali saranno sottoposti apoptosi. </li></ol></li><li> Al momento della formazione del tubo di picco, i media accuratamente aspirato dal pozzo per evitare di interrompere la rete tubo sul BME. <ol><li> Lavare ogni pozzetto con 1 ml DPBS, quindi aspirarla. <ol><li> Per la visualizzazione immediata, aggiungere 1 ml DPBS freschi a ciascuna rete bene e l&#39;immagine del tubo utilizzando un microscopio invertito collegato a una macchina fotografica digitale, o fluorescenza / microscopio confocale se le cellule sono state colorate con calceina AM come in 4.1.1. </li><li> Per fissare il tubo di rete per la visualizzazione successiva, e aggiungere 4% paraformaldeide per ogniaspirato pozzetto ed incubare per 15 minuti a temperatura ambiente. <ol><li> Rimuovere paraformaldeide, lavare due volte con DPBS, quindi aggiungere 1 ml DPBS fresche per ogni bene. </li><li> Microscopicamente immagine prima della fissazione come alcuni tubi possono rompersi durante questa procedura. </li></ol></li></ol></li></ol></li></ol> 6 Quantificazione del tubo Network <ol><li> Quantificare la rete metropolitana in diversi modi a seconda delle preferenze del ricercatore, e diversi programmi di computer hanno capacità di misurare i seguenti parametri: numero di tubi; numero di cicli / maglie; numero di siti di filiale / nodi; lunghezza dei tubi. </li><li> Utilizzare il programma di computer rappresentativa come J Immagine con il plugin Angiogenesi Analyzer 16 per la quantificazione delle reti di tubi. </li></ol>

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L&#39;angiogenesi è coinvolta nei processi sia fisiologici e patologici. Studiare i meccanismi coinvolti nell&#39;angiogenesi richiede l&#39;uso di saggi che ricapitolano le principali fasi di angiogenesi. Il tubo cellule saggio di formazione endoteliali offre diversi vantaggi rispetto ad altri saggi. E &#39;facile set-up, è relativamente poco costoso, produce tubuli in poche ore, ed è quantificabile. Inoltre, esso può essere completato in 24 o 96 pozzetti piastre, e quindi può essere utilizzato per lo screening ad...

L&#39;angiogenesi, lo sviluppo di nuovi vasi sanguigni da vasi preesistenti, è di vitale importanza per una varietà di processi tra cui la crescita di organi, lo sviluppo embrionale, e la guarigione delle ferite 1-3. Recentemente ha sviluppato i vasi sanguigni, rivestiti da cellule endoteliali, l&#39;apporto di ossigeno e sostanze nutritive ai tessuti, promuovere la sorveglianza immunitaria da parte delle cellule ematopoietiche e rimuovere i prodotti di scarto 2,4. L&#39;angiogenesi è di fondamentale importanza durante lo sviluppo embrionale e fetale. Tuttavia, questo processo rimane latente negli adulti eccetto durante i periodi di guarigione delle ferite, la crescita scheletrica, la gravidanza o durante il ciclo mestruale 1-3. Negli ultimi due decenni, i meccanismi molecolari fondamentali che regolano l&#39;angiogenesi hanno cominciato ad emergere. L&#39;angiogenesi è un evento strettamente regolamentato, equilibrato per segnali pro e anti-angiogenici, tra cui integrine, chemochine, angiopoietine, agenti di rilevamento ossigeno, molecole giunzionali e inibitori endogeni 5. Una volta proangsegnali iogenic come la base il fattore di crescita dei fibroblasti (bFGF), fattore di crescita vascolare endoteliale (VEGF), fattore di crescita derivato piastrine (PDGF), e il fattore di crescita epidermico (EGF) attivano cellule endoteliali dei recettori delle cellule endoteliali rilasciano proteasi a degradare la membrana basale. Le cellule endoteliali poi proliferano e migrano, formando germogli a una velocità di alcuni millimetri al giorno 6,7. L&#39;angiogenesi è associata a varie condizioni patologiche, tra cui il cancro, la psoriasi, la retinopatia diabetica, artrite, asma, malattie autoimmuni, malattie infettive, e l&#39;aterosclerosi 8-10. Data l&#39;importanza dell&#39;angiogenesi in varie malattie, la comprensione dei geni e vie che regolano questo processo è fondamentale per la progettazione di terapie migliori. Il saggio di formazione del tubo è un metodo rapido e quantitativo per la determinazione geni o meccanismi coinvolti nell&#39;angiogenesi. In primo luogo descritto nel 1988,i principi alla base di questa analisi sono che le cellule endoteliali mantengono la capacità di dividersi e migrare rapidamente in risposta ai segnali angiogenici 11-13. Inoltre, le cellule endoteliali vengono indotte a differenziarsi e formare strutture tubo-come quando coltivate su una matrice di estratto di membrana basale (BME). Questi tubi contengono un lume circondato da cellule endoteliali collegate tra loro attraverso complessi giunzionali. Formazione del tubo avviene rapidamente con la maggior parte dei tubi che formano in questo test entro 2-6 ore a seconda della quantità e del tipo di stimoli angiogenici. Diversi tipi di cellule endoteliali possono essere usati per questo dosaggio compreso sia cellule primarie e linee cellulari immortalizzate 14,15. La linea cellulare utilizzata per questo articolo è stata del mouse 3B-11, ma la stessa metodologia può essere applicata con le altre linee di cellule endoteliali, come SVEC4-10 (topo) o cellule endoteliali primarie come le cellule umane (HUVEC). A seconda di quale linea cellulare è usato e se ce endotelialeLLS sono trasformati o non trasformati, l&#39;ottimizzazione dovrà essere effettuata per identificare il momento ideale necessaria per la corretta formazione del tubo.

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			<td height="114" rowspan="2" style="height:114px;width:339px;">Costar 24-Well Tissue Culture-Treated Plate</td>
			<td rowspan="2" style="width:189px;">Corning</td>
			<td rowspan="2" style="width:124px;">3524</td>
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			<td height="190" rowspan="2" style="height:190px;width:339px;">BD Matrigel Basement membrane matrix, Growth factor reduced</td>
			<td rowspan="2" style="width:189px;">BD Biosciences</td>
			<td rowspan="2" style="width:124px;">354230</td>
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			<td height="95" rowspan="2" style="height:95px;width:339px;">Gibco L-Glutamine 200mM (100X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25030-081</td>
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			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco Pen Strep</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">15140-122</td>
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			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DMEM (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">11965-092</td>
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			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DPBS (1X)</td>
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			<td rowspan="2" style="width:189px;">Atlas Biologicals</td>
			<td rowspan="2" style="width:124px;">F-0500-A</td>
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			<td height="21" style="height:21px;width:339px;">Calcein AM</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:124px;">C3100MP</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMSO</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:124px;">4-X</td>
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endothelial cell tube formation assay for the in vitro study of angiogenesis

L&#39;angiogenesi è un processo fondamentale per lo sviluppo del tessuto normale e la guarigione della ferita, ma è anche associato con una varietà di condizioni patologiche. Usando questo protocollo, l&#39;angiogenesi può essere misurata in vitro in un veloce, modo quantificabile. Cellule endoteliali primarie o immortalati sono mescolati con i media condizionati e placcato su matrice membrana basale. Le cellule endoteliali formano strutture simili a capillari in risposta ai segnali angiogenici trovati in mezzi condizionati. La formazione del tubo avviene rapidamente con le cellule endoteliali cominciando ad allinearsi entro 1 ora e tubuli-lume contenente cominciando ad apparire in 2 ore. I tubi possono essere visualizzati con un contrasto di fase invertito microscopio, o le cellule possono essere trattate con calceina AM prima del test e tubi visualizzati tramite fluorescenza o confocale. Il numero di siti di filiale / nodi, loop / mesh, o il numero o la lunghezza dei tubi formati può essere facilmente quantificato in una misura di in vitro angiogenesi. In sintesi, questo test può essere utilizzato per identificare i geni e vie che sono coinvolti nella promozione o inibizione dell&#39;angiogenesi in modo rapido, riproducibile, e quantitativa.

Mouse 3B-11 cellule endoteliali sono state seminate su solidificato fattore di crescita ridotta BME - in questo saggio, il prodotto Matrigel è stato utilizzato - e seguito nel tempo. Come mostrato in Figura 1, fattori angiogenici secreti da entrambi cheratinociti o fibroblasti di topo sono capaci di indurre la formazione del tubo nel tempo. Le cellule endoteliali migrano e cominciano a formare piccoli rami in 1-2 ore di placcatura. Formazione massima è stato raggiunto 4-6 ore utilizzando mezzi condizi...

Watch this Scientific Journal Video about Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis at JoVE.com

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Il saggio di formazione del tubo è un metodo veloce e quantificabili per misurare l&#39;angiogenesi in vitro. Le cellule endoteliali sono combinati con i media condizionati e piastrate su estratto di membrana basale. Formazione del tubo avviene entro ore e tubuli appena formati facilmente quantificabile.

Endoteliali Tubo cellulare saggio Formazione per la<em&gt; In Vitro</em&gt; Studio di Angiogenesi

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

59.0K Views.  American University.  Il saggio di formazione del tubo è un metodo veloce e quantificabili per misurare l'angiogenesi in vitro. Le cellule endoteliali sono combinati con i media condizionati e piastrate su estratto di membrana basale. Formazione del tubo avviene entro ore e tubuli appena formati facilmente quantificabile. 

Video: Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

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Biologia

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes: un modello in vitro per lo studio dell&#39;angiogenesi (Intervista)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Ottimizzato fibrina Gel Bead Assay per lo studio della angiogenesi

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Un Matrigel-Based Formazione Assay tubo per valutare l&#39;attività vascolare delle cellule tumorali

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositivo per il recupero di campioni crioconservati cadere accidentalmente nelle cisterne criogeniche

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Rat Esteriorizzazione mesentere: un modello per indagare le dinamiche cellulari coinvolti nell&#39;angiogenesi

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

La quantificazione di adesività delle cellule endoteliali<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

Un saggio biologico semplice per la valutazione dei fattori di crescita dell&#39;endotelio vascolare

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.