DIY criogenico dispositivo di recupero era stato testato in corso dei progetti &quot;prenilazione proteica Modeling batteriche nelle cellule eucariotiche usando la bioinformatica e approcci molecolari&quot;, promosso dal Premio Mary Louise Andrews per il programma di ricerca sul cancro, Virginia Academy of Science &quot;, significato fisiologico di RNA editing in Plasma Le cellule dendritiche &quot;promosso dal Jeffress F. Thomas e Kate Miller Jeffress Memorial Trust e contratto 16.740.11.0364 Stato (Ministero della Scienza e Istruzione, Russia). Vorremmo ringraziare Beth Eom per l&#39;assistenza con l&#39;esperimento integrità RNA.

1. Montaggio del dispositivo per il recupero di Cryotolerant Cryoboxes <ol><li> Utilizzando pinza, raddrizzare un lato di 3 lati forte legame (Figura 1A) per effettuare una forma di L-forte legame nota mostrata in figura 1B:. Le dimensioni del forte legame-può essere selezionata a seconda del diametro del collo di Dewar . </li><li> Fissare due tali forte legame con un forte legame cinghia T (figura 2) utilizzando corona dado del bullone, la rondella e la vite per formare la base Cryoscoop come mostrato in Figura 3 (AB) Nota:. Due legami forti vengono utilizzati per consentire il recupero dei 5 ½ pollici X 5 ½ pollici cryoboxes. </li><li> Utilizzando una piastra scanalata (figura 4), ​​allineare le aperture sul forte legame cinturino T con le scanalature su un&#39;estremità della piastra asolata e fissare i due insieme con l&#39;aiuto di dado Hillman e bulloni (Figura 5). Nota: Se la piatto forato e forte legame non si come con fori predisposti, si dovrà avere fori praticati con punta in metallo duro. </li><li> Piegare l&#39;altra estremità della piastra asolata con l&#39;aiuto di una pinza come mostrato in Figura 6. (Questo passaggio è facoltativo). </li><li> Avvolgere la maniglia con schiuma di comprendere meglio il dispositivo quando in azoto liquido, come mostrato nella Figura 6. (Opzionale) Nota: rimuovere la schiuma prima della sterilizzazione in autoclave. </li></ol> 2. Montaggio del dispositivo per il recupero di Cryotolerant Cryovials Il Cryotolerant dispositivo di recupero fai da te può essere adattato anche per il recupero di cryovials. Ciò comporta sostituire la base piana con un filtro in acciaio inossidabile come mostrato alterato per scavando flaconi da azoto liquido (Figura 7). <ol><li> Svitare i dadi della Corona e bulloni che fissano il forte legame cinghia T alla piastra asolata. </li><li> Prima di applicare il filtro in acciaio inox alla piastra asolata, flettere leggermente il colloil filtro con l&#39;aiuto di pinze per orientare la base ad un angolo per assomigliare ad una siviera (Figura 8) Nota: Se il filtro è dotato di un labbro, utilizzare una pinza per piegare il labbro basso, questo renderà manovre con il dispositivo più facile.. </li><li> Allineare il foro sul manico del filtro con i fori sulla piastra asolata e fissare il filtro con l&#39;aiuto di Hillman dadi e bulloni, come mostrato nella Figura 8. Nota: se il filtro non viene fornito con fori predisposti, sarà necessario di organizzare per avere fori con la punta in metallo duro. </li></ol> 3. Recupero Cryoboxes e Cryovials Da Dewar <ol><li> Per iniziare, indossare guanti criogenici e togliere 1 o 2 rack freezer dal Dewar per lasciare spazio di manovra. </li><li> Raschiare delicatamente lungo la parte inferiore del Dewar con il montante Cryotolerant dispositivo di recupero fai da te dotata di una forte base di strap tie-T (Figura 5) e raccogliere l&#39;Falle boxn al fondo del serbatoio Dewar Nota:. bolle azoto liquido immediatamente a contatto con un oggetto più caldo, avvolgente l&#39;oggetto in isolante gas azoto (effetto Leidenfrost). Per raffreddare il dispositivo di recupero prima di campioni, lentamente immergere il dispositivo nella Dewar e consentire azoto liquido per raffreddare il dispositivo sufficientemente prima di procedere con la fase precedente. </li><li> Tirare il dispositivo in posizione verticale lungo le pareti del Dewar per raccogliere la scatola freezer dal serbatoio. </li><li> Afferrare la scatola recuperata dal fondo della Dewar. </li><li> Per il recupero cryovials, delicatamente raschiare il fondo del Dewar con la cryoscoop munito di un filtro (Figura 9). </li><li> Simile a cryobox recupero, estrarre il dispositivo in posizione verticale e prendere le fiale raccolti nel filtro. </li></ol> 4. Risultati rappresentativi Una sfida in studi di espressione genica è che la qualità dell&#39;RNA dipende dalle condizioni di conservazioneper entrambi i campioni di tessuto congelato e la Figura estratto RNA 4. 10 mostra l&#39;effetto della manipolazione del campione sull&#39;integrità di RNA totale. L&#39;RNA totale dalla sorgente stesso tessuto (adiposo umano) è stato sottoposto a manipolazione dei campioni differenti. Prima di esperimento, intatto RNA totale preparato mostra chiare bande 28S e 18S rRNA con la banda 28S rRNA approssimativamente due volte più intenso come la banda 18S rRNA (Figura 10, corsia 1). Questo rapporto 2:1 (28S: 18S) è un&#39;indicazione che l&#39;RNA è intatto, mentre parzialmente degradata RNA appare come una macchia, o la sua 28S: intensità 18S rapporto parte da 2:01 5. Lanes 3-4 raffigura campioni di RNA con una degradazione significativa. Questi campioni di RNA sono stati sottoposti a ripetuti scongelamento parziale di breve termine richiami il Dewar (metodo A), modellando così stoccaggio a lungo termine con periodica decantazione a causa della necessità di recuperare il fiale caduta e caselle (Figura 10, corsia 3-4 ). Tuttavia, i campioni conservati in Dewar servitoutilizzare un dispositivo DIY (Metodo B) e, quindi, non scongelamento parziale, contengono RNA intatto (Figura 10, corsia 2). <img alt="Figura 1" src="/files/ftp_upload/3903/3903fig1.jpg" /> Figura 1. A) La base è costituito da due tre lati dimensioni indicate forte della cravatta. Ogni forte legame contiene una grande pre-forate per infilare (freccia solido) e fori più piccoli (più piccola freccia) su ciascuna faccia. Questo fornisce un opzione per consentire il fissaggio con dado e bulloni. Anche i fori più piccoli consentono il rapido deflusso del liquido durante il recupero del campione. B) Un lato di ogni della forte legame viene raddrizzata con l&#39;aiuto di una pinza per rendere L sagomato forte legame. <img alt="Figura 2" src="/files/ftp_upload/3903/3903fig2.jpg" /> Figura 2. Strong-tie strap T viene utilizzato per proteggere i forti legami di insieme. Come forte legame, T-strap ha una grande pre-forate per infilare (freccia piena) e due fori più piccoli (SMALl freccia) in ogni angolo. Questo permette di fissaggio forte legame per tutta la lunghezza del T - Strap con viti e bulloni. Consente inoltre di fissaggio T-strap alla piastra asolata. <img alt="Figura 3" src="/files/ftp_upload/3903/3903fig3.jpg" /> Figura 3. A) Ogni forte legame è fissata in un angolo di T-strap, tutta la sua lunghezza con i dadi dei bulloni corona, bulloni e rondelle. B) Due forte di legami sono fissati uno accanto all&#39;altro per aiutare recuperare 5 ½ X 5 ½ pollici dentro cryoboxes. Per i più piccoli cryoboxes come 2 mm x 2 pollici, un unico forte legame può essere fissato direttamente alla piastra asolata senza T-strap. <img alt="Figura 4" src="/files/ftp_upload/3903/3903fig4.jpg" /> Figura 4. Intaglio piastra piatta viene utilizzato come impugnatura. Lunghezza della piastra asolata dipende dalla profondità della Dewar. In questo caso, 4 piedi di piastra asolata stato utilizzato. Lo spessore della piastra scanalata s è criticacia sottili lastre scanalate (&lt;14 calibri) rendere il dispositivo fragile durante il recupero di campioni di azoto liquido riempito Dewar. <img alt="Figura 5" src="/files/ftp_upload/3903/3903fig5.jpg" /> Figura 5. La base piana costituita da forti legami di cinghia e T è poi fissato ad una estremità della piastra asolata. Il buco più grande all&#39;angolo rimanente della cinghia T è allineata con la fessura sulla piastra piatta e fissata con bulloni e dado esagonale. Questo dispositivo può essere ora utilizzato per recuperare cryoboxes da Dewar. <img alt="Figura 6" src="/files/ftp_upload/3903/3903fig6.jpg" /> Figura 6. Una estremità della piastra scanalata piana è piegata con una pinza e ricoperto di poliuretano per fornire una migliore aderenza soprattutto per Dewars profonde, come mostrato in questo caso. <img alt="Figura 7" src="/files/ftp_upload/3903/3903fig7.jpg" /> Figura 7. Filtro con rete metallica e maniglia in acciaio inoxviene utilizzato per recuperare cryovials da Dewar. Se la maniglia non è provvisto di una scanalatura, con trapano bit carburo saranno necessari per la perforazione di un foro. <img alt="Figura 8" src="/files/ftp_upload/3903/3903fig8.jpg" /> Figura 8. Per facilitare il recupero dei cryovials, filtro viene piegata lungo il collo con una pinza per ottenere filtro a forma di mestolo. Se il filtro è dotato di labbro come in questo caso, pinze possono essere utilizzati per piegarlo. In questo modo il filtro non rimanere intrappolati all&#39;interno della Dewar. <img alt="Figura 9" src="/files/ftp_upload/3903/3903fig9.jpg" /> Figura 9. Filtro è fissata alla piastra asolata piatta ad una estremità con dado e bullone. <img alt="Figura 10" src="/files/ftp_upload/3903/3903fig10.jpg" /> Figura 10. L&#39;integrità del campione di RNA totale sottoposta a diverse condizioni di gestione dei campioni. Per esplorare come la qualità RNA è influenzato dal campione gestione condizioni, una serie di campioni di RNA dalla stessa fonte è stato sottoposto a due metodi di manipolazioni. Metodo A consisteva di stoccaggio di RNA in Dewar sottoposto a recuperi di esempio utilizzando dispositivo fai da te, quindi non scongelamento parziale. Metodo B consisteva di stoccaggio di RNA in Dewar sottoposto a ripetute scongelamento parziale a breve termine richiami effettuati nell&#39;ambito del Dewar, modellando così una conservazione a lungo termine con travasi periodici per il recupero dei campioni caduti. Integrità RNA totale è stato poi analizzato mediante elettroforesi su gel. Lane1: campione prima dello stoccaggio in Dewar; Lane 2: esempio da Dewar sottoposto a recupero secondo il metodo A; Lane 3, 4: Esempio da Dewar sottoposti a recupero con il metodo B, M: ladder 100 bp.

<ol>
	<li>Kittel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Cryogenic Engineering. , 41 (1996).</li><li>Edeskuty, F. J., Walter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stewart Safety in the handling of cryogenic fluids. , (1996).</li><li>Council, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards. , (2011).</li><li>Muyal, J., Muyal, V., Kaistha, B., Seifart, C., Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+RNA+extraction+techniques+from+frozen+and+fresh+lung+tissues:+checkpoint+towards+gene+expression+studies.">Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies.</a> Diagnostic Pathology. 4, 9 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Holland, N. T., Smith, M. T., Eskenazi, B., Bastaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+sample+collection+and+processing+for+molecular+epidemiological+studies.">Biological sample collection and processing for molecular epidemiological studies.</a> Mutation Research/Reviews in Mutation Research. 543, 217-234 (2003).</li><li>Lynch, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CELL,+TISSUE+AND+ORGAN+CULTURE+|+Storage+and+Cryopreservation.">CELL, TISSUE AND ORGAN CULTURE | Storage and Cryopreservation.</a> Encyclopedia of Rose Science. , 106-111 (2003).</li><li>Crawford, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics Engineering. , (1998).</li><li>Gorenc, B., Tinyou, R., Syam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Steel designers' handbook. , (1996).</li><li>Harper, C. A., Petrie, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastics+materials+and+processes:+a+concise+encyclopedia.">Plastics materials and processes: a concise encyclopedia.</a> Wiley-Interscience. , (2003).</li></ol>

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Uno dei metodi più comuni di recupero cryoboxes e cryovials dal fondo del serbatoio Dewar è decantare azoto liquido in un contenitore di riserva ed estrarre i campioni rimanenti nel pallone o galleggiare in azoto liquido decantato. Questo è, tuttavia, una pratica sicura, che può provocare ustioni criogeniche o di asfissia a causa di una prolungata esposizione ai vapori di azoto liquido 2. Altro modo comune per il recupero dei caduti cryovials comporta l&#39;uso di pinze esageratamente 3. Tuttav...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Hillman Group placcato intaglio piatto 1-3/8 x 4 metri; 14 Gauge </td><td> Hardware negozio locale </td><td> N / A </td><td> 1 unità necessaria </td></tr><tr><td> Crown Bolt Stainless Steel # 6 - 32 x 3/8 in Oval Screw Machine Head unità Phillips </td><td> Hardware negozio locale </td><td> Model # 42281 </td><td> 4 unità richieste </td></tr><tr><td> Bolt Crown 3/8 pollici x 1-1/2 in rondelle acciaio zincato Fender </td><td> Hardware negozio locale </td><td> Model # 20122 </td><td> 4 unità richieste </td></tr><tr><td> Crown Bolt zincati # 8-32 Vite Dado Macchina </td><td> Hardware negozio locale </td><td> Model # 18512 </td><td> 4 unità richieste </td></tr><tr><td> Simpson Strong-Tie BC40Z 4 pollici In.x4 Mezza Base </td><td> Hardware localeacquisti </td><td> Model # BC40Z </td><td> 2 unità richieste </td></tr><tr><td> Simpson Strong-Tie 6 pollici x 6 pollici Strap T </td><td> Hardware negozio locale </td><td> Model # 66T </td><td> 1 unità necessaria </td></tr><tr><td> Oneida 6 &quot;Mesh Strainer </td><td> Supermercato locale </td><td> Model # 7859W </td><td> 1 unità necessaria </td></tr><tr><td> Il Gruppo Hillman zincato Hex Nuts 3/8-16 </td><td> Hardware negozio locale </td><td> Model #: 810509 </td><td> 6 unità richieste </td></tr><tr><td> Il Gruppo Hillman zincato bulloni esagonali 3/8- 16 </td><td> Hardware negozio locale </td><td></td><td> 4 unità richieste </td></tr><tr><td> Espanso per l&#39;isolamento </td><td> Hardware negozio locale </td><td></td><td> 1 unità necessaria </td></tr><tr><td> Cacciavite </td><td> Hardware negozio locale </td><td></td><td> 1 unità necessaria </td></tr><tr><td> Pinze </td><td> Hardware negozio locale </td><td></td><td> 1 unità necessaria </td></tr></tbody></table>

do-it-yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

L&#39;azoto liquido è incolore, inodore, estremamente freddo (-196 ° C) di liquido mantenuto sotto pressione. È comunemente usato come fluido criogenico per conservazione a lungo termine di materiali biologici come sangue, cellule e tessuti 1,2. La natura criogenico di azoto liquido, mentre l&#39;ideale per la conservazione del campione, può causare congelamento rapido dei tessuti vivi a contatto - noto come &#39;criogenico burn&#39; 2, che possono portare a gravi congelamenti in persone strettamente coinvolte nella conservazione e il recupero dei campioni di Dewars. Inoltre, come azoto liquido evapora riduce la concentrazione di ossigeno nell&#39;aria e possono causare asfissia, specialmente in spazi ristretti 2. Nei laboratori, i campioni biologici sono spesso conservati in cryovials cryoboxes o impilati in rack in acciaio inox all&#39;interno dei serbatoi Dewar 1. Questi scaffali sono provvisti di un albero lungo per evitare scatole di scivolare fuori dai rack e sul fondo dellaDewars durante la routine di gestione. Troppo spesso, tuttavia, scatole o fiale con i campioni preziosi scivolare fuori e si depositano sul fondo del serbatoio di azoto liquido riempito. In tali casi, i campioni possono essere tediously recuperato dopo il trasferimento della azoto liquido in un contenitore di riserva o eliminazione. Le scatole e flaconi possono essere relativamente sicuro recuperato dal svuotato Dewar. Tuttavia, la natura criogenico di azoto liquido e il suo tasso di espansione rende pericolosa affondata il recupero del campione. È comunemente raccomandato da Uffici sicurezza che il recupero del campione non essere effettuata da una sola persona. Un&#39;altra alternativa è quella di utilizzare grabber fresche reperibili in commercio o pinze per estrarre i flaconi 3. Tuttavia, la visibilità limitata all&#39;interno degli oscuri Dewars pieni di liquido costituisce un grande limite nel loro utilizzo. In questo articolo, si descrive la costruzione di un dispositivo di recupero Cryotolerant fai da te, che rende il recupero del campione da Dewar contenente liquidi criogenici sia sicuro unond facili.

Watch this Scientific Journal Video about Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks at JoVE.com

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

Presentiamo qui un basso costo, durevole cryotolerant dispositivo per il recupero del campione da serbatoi Dewar riempiti con azoto liquido. La facilità di costruzione e la progettazione modulare del dispositivo rende il processo di recupero campione da serbatoi criogenici sicuro e facile.

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 ...

do-it-yourself-device-for-recovery-cryopreserved-samples-accidentally

Research

JoVE Journal

Biology

25.2K Views.  George Mason University. Presentiamo qui un basso costo, durevole cryotolerant dispositivo per il recupero del campione da serbatoi Dewar riempiti con azoto liquido. La facilità di costruzione e la progettazione modulare del dispositivo rende il processo di recupero campione da serbatoi criogenici sicuro e facile.

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

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabbricazione di un dispositivo a microfluidi per la compartimentazione dei Neuron Soma e assoni

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Ricerca

Biologia

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Un gradiente di generazione del dispositivo Microfluidic di Biologia Cellulare

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Il trapianto di cellule direttamente nel rene di Zebrafish adulti

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

I campioni di prova per ottimizzare STORM Super-Resolution Microscopia

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Analisi metagenomica quasi di Salmonella da campioni ambientali e alimentari

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Tecniche riproduttive per il monitoraggio e il controllo ovarico negli anfibi

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Caricamento criogenico del campione in uno spettrometro a risonanza magnetica nucleare ad angolo magico che preserva la vitalità cellulare

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Imaging privo di etichette delle dinamiche di conservazione lipidica negli elegani di caenorhabditis utilizzando la microscopia a dispersione Raman stimolata

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.