Noi riconosciamo con gratitudine fondi avvio da Wayne State University (MJA), una borsa di studio dalla Fondazione Americana per Aging Research (SMV), e un percorso di transizione Career Award Indipendenza (R00EB007129) dal National Institute of Biomedical Imaging e Bioingegneria del National Institutes della Sanità (MJA).

1. Metalation utilizzando LnCl 3 sali <ol><li> Sciogliere il ligando in acqua per produrre una soluzione di 30-265 mM. Il ligando 2 - (4-isothiocyanatobenzyl)-dietilentriammina acido pentaacetic (p-SCN-Bn-DTPA) è stato utilizzato in questo video in una concentrazione di 73 mm. </li><li> Regolare il pH della soluzione di ligando compreso tra 5,5 e 7,0 con l&#39;aggiunta di 1 M NH 4 OH. In questo video, 0,2 ml di M 1 soluzione di NH 4 OH è stato utilizzato. </li><li> Sciogliere 1-2 equivalenti di LnCl 3 in acqua per produrre una soluzione con una concentrazione di 5.000-1.000 mM. In questo video, EuCl GdCl 3 e 3 sono stati usati in concentrazioni di 111 mm. Un eccesso di metallo è spesso usato per guidare il metalation di completamento e di conseguenza semplificare purificazione. </li><li> Aggiungere la soluzione di LnCl 3 per la soluzione del ligando mescolando. </li><li> Dopo l&#39;aggiunta di LnCl 3, regolare il pH della miscela di reazione risultante tra 5,5 e 7,0 con l&#39;aggiunta di 0,2 M di NH 4 OH. Un totale di 0,5 ml di 0,2 M soluzione di NH 4 OH è stato utilizzato in questo video. Se il legante contiene gruppi funzionali sensibili agli acidi, regolare il pH più volte durante questa fase. ATTENZIONE - Se la soluzione diventa troppo di base, tutti i gruppi di base sensibili funzionali, come isotiocianato, sarà reso inutilizzabile per coniugazione. </li><li> Monitorare la reazione attraverso misure di pH. La reazione è completa quando il pH rimane costante. </li></ol> 2. PH iter Raising (non incluso in questo video, ma buona per ligandi senza gruppi funzionali di base e minuscole) <ol><li> Aggiungi concentrato NH 4 OH alla miscela di reazione per regolare il pH a ≥ 11. Questo passo precipitare qualsiasi metallo uncomplexed come idrossido insolubile. </li><li> Filtrare il surnatante attraverso un filtro 0,2 micron. Se la miscela di reazione intasa il filtro, centrifugazione e decantazione prima di filtraggio è raccomandato. </li><li> Se la dialisi non verrà eseguita, rimuovere il solvente a pressione ridotta (evaporazione rotante o liofilizzazione è consigliato). </li><li> Passi 2,1-2,3 può essere ripetuto se lantanidi libero rimane. </li></ol> 3. Dialisi workup <ol><li> Tagliare il tubo dialisi ad una lunghezza appropriata (seguire le linee guida del produttore) per tenere il volume del campione, lasciando lunghezza in più (circa il 10% del volume del campione). In questo video, un 100-500 dalton peso molecolare di cut-off (MWCO) membrana è stata utilizzata, ma più grandi tubi MWCO può essere utilizzato come coniugazione appropriata se viene eseguita prima di metalation. Inoltre, le cassette dialisi può essere utilizzato come alternativa ai tubi di dialisi se lo si desidera. </li><li> Se del caso sulla base di linee guida del produttore, immergere il tubo dialisi taglio in acqua per 15 minuti a temperatura ambiente. </li><li> Riempire un serbatoio di dialisi (1 bicchiere L è stato utilizzato in questo video) con l&#39;acqua (dialisato). Il volume dialisato dovrebbe essere di circa 100 volte quella del campione. </li><li> Piegare un&#39;estremità del tubo due volte e fissare la parte piegata del tubo con una fascetta di chiusura dialisi. Avvolgere la fine della chiusura con un elastico per garantire che rimanga chiuso durante la dialisi. </li><li> Filtrare la miscela di reazione attraverso un filtro 0,2 micron, e caricare il filtrato nella parte aperta del tubo facendo attenzione a non strappare il tubo. Assicurarsi di lasciare sufficiente spazio testa per chiudere il tubo. </li><li> Piegare l&#39;altra estremità aperta del tubo due volte, sicuro con una chiusura, e avvolgere la chiusura con un elastico come al punto 3.4. </li><li> Collegare un flaconcino di vetro contenente aria al morsetto su una delle estremità del tubo dialisi con un elastico. Fissare un flacone contenente sabbia per l&#39;altro morsetto. Queste fiale in modo che il tubo rimane immerso nel dializzato. </li><li> Posizionare il tubo pieno nel serbatoio che contiene dialisi dialisi. </li><li> Mescolare il dializzato utilizzando una piastra magnetica mescolare a bassa velocità (non vortex) a temperatura ambiente. </li><li> Modificare il dialisato 3x nel corso di una giornata (in questo video, il dialisato è stato cambiato a 2,5, 6,5, e 11,5 ore), e quindi consentire di continuare la dialisi durante la notte (per un totale di 20-28 ore di dialisi). </li><li> Rimuovere il tubo di dialisi dal dialisato e attentamente aprire una chiusura a rimuovere l&#39;esempio. Lavare il circuito di dialisi 3x con acqua e combinare i lavaggi con il campione. </li><li> Rimuovere l&#39;acqua a pressione ridotta. Liofilizzazione è utilizzato in questo video. </li></ol> 4. Valutazione della presenza di metallo libero <ol><li> Sciogliere il complesso metallico a tampone (buffer di preparazione: Sciogliere 1,4 ml di acido acetico in 400 mL di acqua, aggiustare il pH a 5,8 con 1 M NH 4 OH, e aggiungere acqua per produrre un volume totale di 500 mL) e aggiungere il xilenolo Indicatore arancione (16 mM in tampone pH 5,8). In questo video, 0,3 mg di complesso è stato sciolto in 0,3 ml di buffer e 3 ml di soluzione d&#39;indicatore è stato aggiunto. </li><li>Rilevare la presenza di metallo libero attraverso l&#39;osservazione di un cambiamento di colore dell&#39;indicatore dal giallo al viola. </li><li> Se lo si desidera, la quantità di metallo libero può essere quantificata mediante la creazione di una curva di calibrazione 1. In alternativa, il colorante arsenazo III può essere usato al posto di xilenolo arancio 2. Se il metallo libero rimane, il campione deve essere ulteriormente purificato tramite dialisi, una colonna di dissalazione, o ad alte prestazioni cromatografia liquida (HPLC) prima caratterizzazione. </li></ol> 5. Determinazione di acqua coordinamento numero (q) <ol><li> Preparare una soluzione del Eu III contenenti complesso (~ 1 mm) in H 2 O e un&#39;altra soluzione della stessa concentrazione in D 2 O. Prima dell&#39;analisi, la soluzione D 2 O devono essere evaporata e sciolto in D 2 O tre volte per rimuovere residui di H 2 O. </li><li> Aggiungere la soluzione di acqua ad una cuvetta pulita, e posizionare la cuvetta in una spettrofluorimetro. </li><li> Eseguire scansioni di eccitazione ed emissione per determinare i massimali previsti per ogni (~ ~ 395 nm e 595 nm, rispettivamente). </li><li> Eseguire una fosforescenza tempo di decadimento esperimento utilizzando i seguenti parametri: lunghezza d&#39;onda di eccitazione e di emissione stabiliti a partire dal punto 5.3, eccitazione e fenditure emissione (5 nm), flash contano (100), ritardo iniziale (0,01 ms), ritardo massimo (13 ms) e incremento di ritardo (0,1 ms). Queste condizioni sono adatte alla maggior parte dei complessi, ma il ritardo massimo e valori di incremento può essere aumentata o diminuita per le specie con tempi di decadimento molto lunghi o molto corti. </li><li> Ripetere il passo 5,4 con la soluzione di D 2 O preparato al punto 5.1. </li><li> Dal luminescenza-decadimento dati ottenuti in 5.4 e 5.5, la trama il logaritmo naturale di intensità in funzione del tempo. La pendenza di queste linee sono i tassi di decadimento (τ -1) (Figura 2). In questo video, Microsoft Excel 2007 è stato utilizzato per generare le trame logaritmo naturale dai dati grezzi. Utilizzare i tassi di decadimento nella equazione sviluppata da Horrocks e collaboratori (eq 1) 3. Se il legante contiene gruppi OH o NH coordinati al metallo, allora l&#39;equazione deve essere modificata prima dell&#39;uso 3. </li></ol> eq 1: <img src="/files/ftp_upload/2844/2844eq1.jpg" alt="Equazione 1" /> 6. Relassività misure <ol><li> Selezionare la modalità di applicazione desiderata sull&#39;analizzatore tempo di rilassamento: T 1 (tempo di rilassamento longitudinale) o T 2 (tempo di rilassamento trasversale). </li><li> Preparare una serie di campioni che contengono diverse concentrazioni di Gd III contenenti complessi in un solvente acquoso. In questo video, l&#39;acqua è stato utilizzato come solvente e soluzioni di 10.0, 5.00, 2,50, 1,25, 0,625 e 0 mm sono stati preparati. Altri solventi acquosi o buffer potrebbe essere usato, ma è importante utilizzare il solvente come il vuoto. Il volume finale del campione è specifico per lo strumento che viene utilizzato. </li><li> Porre un campione nello strumento e lasciare riposare per 5 minuti per equilibrare la temperatura dello strumento (37 ° C in questo video). </li><li> Determinare il tempo di rilassamento (in unità di s) regolando i parametri del software per ottenere una curva esponenziale per T 1 o T 2 (curve rappresentante per T 1 e T 2 sono mostrati in Figura 3). </li><li> Ripetere i passi 6.3 e 6.4 per tutti i campioni compreso il bianco. </li><li> Calcolare l&#39;inverso della misurato T 1 o T 2 valori in unità di s -1. </li><li> Tracciare la T o T 1 -1 2 -1 valori in funzione della concentrazione Gd III (in unità di mM). A causa della natura igroscopica di Gd III contenenti complessi, confermare la concentrazione di Gd III con spettrofotometria ad assorbimento atomico o spettrometria di massa ad accoppiamento induttivo plasma. Montare la trama con una linea retta. Un diagramma rappresentante è mostrata in Figura 4. </li><li> La pendenza della retta è la relassività (r 1 e r 2 per T 1 e T 2, rispettivamente) ed ha unità di mM -1 s -1. </li></ol> 7. Rappresentante Risultati Dati rappresentativi per i passaggi di questo protocollo sono stati inseriti nelle tabelle e figure sezione. Oltre all&#39;acqua coordinamento numero e la caratterizzazione relassività descritte nel protocollo, è importante per caratterizzare prodotti finali utilizzando tecniche chimiche standard. L&#39;identità del composto può essere ottenuto utilizzando la spettrometria di massa, spettri di massa e di rappresentanza che mostra i modelli isotopo diagnostici per Gd III - III e Eu contenenti complessi sono mostrati in figura 5. Inoltre, per i non-Gd III </sfino&gt; lantanidi contenenti complessi, spettroscopia NMR può essere utilizzata per l&#39;identificazione del prodotto. Per caratterizzare la purezza del complesso, un&#39;analisi HPLC o elementare o entrambe possono essere usate. <img src="/files/ftp_upload/2844/2844fig1.jpg" alt="Figura 1" /> Figura 1 Schema generale per metalation e purificazione:. Schema raffigurante la procedura generale per metalation e le ragioni per la scelta di percorsi diversi purificazione. <img src="/files/ftp_upload/2844/2844fig2.jpg" alt="Figura 2" /> Figura 2 luminescenza intensità trama:. Trama Rappresentante del logaritmo naturale di intensità in funzione del tempo dalla sezione 5. Le pendenze delle linee generate da curve simili acquisiti per l&#39;acqua e le soluzioni D 2 O vengono usati con eq 1 a caratterizzare l&#39;acqua coordinamento numero di Eu III contenenti complessi. <img src="/files/ftp_upload/2844/2844fig3.jpg" alt="Figura 3" /> Figura 3 curve di rilassamento tempo di decadimento:. Dati Rappresentante per (a sinistra) T 1 e (a destra) T 2 acquisizione. Deviazioni da queste forme curva dovrebbe produrre dati inattendibili. <img src="/files/ftp_upload/2844/2844fig4.jpg" alt="Figura 4" /> Figura 4 determinazione relassività:. Un grafico rappresentativo di 1 / T 1 contro la concentrazione di Gd III. La pendenza della retta è relassività e dispone di unità di mM -1 s -1. <img src="/files/ftp_upload/2844/2844fig5.jpg" alt="Figura 5" /> Figura 5 spettri di massa:. Spettri di massa rappresentante che mostra i modelli diagnostici per isotopo (a sinistra) Gd III contenenti complessi e (a destra) Eu III contenenti complessi. I picchi neri rappresentano la distribuzione gaussiana teorica isotopo e le linee rosse sono i dati effettivi.

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Nessun conflitto di interessi dichiarati.

Dato il numero crescente di pubblicazioni che includono lantanidi a base di agenti di contrasto 4-14, è importante che la cura nella preparazione, purificazione e caratterizzazione dei prodotti per garantire risultati riproducibili e comparabili. Questi complessi sono spesso considerati difficili da purificare e caratterizzare rispetto alle molecole organiche a causa della loro natura paramagnetica e la sensibilità di gruppi funzionali che potrebbero essere utilizzati per bioconjugation. Abbiamo descritto i...

<table border="1"><tbody><tr><th> Reagenti e attrezzature </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> EuCl 3 • 6H 2 O </td><td> Sigma-Aldrich </td><td> 203254-5G </td></tr><tr><td> p-SCN-Bn-DTPA </td><td> Macrocyclics </td><td> B-305 </td></tr><tr><td> idrossido di ammonio </td><td> EMD </td><td> AX1303-3 </td></tr><tr><td> Spectra / Por Biotech Cellulosa Ester (CE) membrana di dialisi - 500 D MWCO </td><td> Fisher Scientific </td><td> 68-671-24 </td></tr><tr><td> Millipore IC Millex-LG Filtro Unità </td><td> Fisher Scientific </td><td> SLLG C13 NL </td></tr><tr><td> xilenolo arancione sale tetrasodico </td><td> Alfa Aesar </td><td> 41379 </td></tr><tr><td> acido acetico </td><td> Fluka </td><td> 49199 </td></tr><tr><td> D 2 O </td><td> Cambridge Isotope Laboratories, Inc. </td><td> DLM-4-25 </td></tr><tr><td> purificatore d&#39;acqua </td><td> ELGA </td><td> Purelab Ultra </td></tr><tr><td> cromatografia liquida ad alta prestazione e la spettrometria di massa </td><td> Shimadzu </td><td> LCMS-2010EV </td></tr><tr><td> tempo di rilassamento analizzatore </td><td> Bruker </td><td> mq60 minispec </td></tr><tr><td> Spettrofotometro UV-vis </td><td> Fisher Scientific </td><td> 20-624-00092 </td></tr><tr><td> congelare asciugatrice </td><td> Fisher Scientific </td><td> 10-030-133 </td></tr><tr><td> pH-metro </td><td> Hanna Instruments </td><td> HI 221 </td></tr><tr><td> spettrofluorimetro </td><td> HORIBA Jobin Yvon </td><td> Fluoromax-4 </td></tr><tr><td> Molecular Weight Calculator versione 6,46 da Matthew Monroe, scaricato 17 Ottobre 2009 </td><td> <a href="http://ncrr.pnl.gov/software/">http://ncrr.pnl.gov/software/</a> </td><td> Peso Molecolare Calculator </td></tr></tbody></table>

preparation, purification, and characterization of lanthanide complexes for use as contrast agents for magnetic resonance imaging

Polyaminopolycarboxylate a base di leganti sono comunemente utilizzati per chelare ioni lantanidi, ed i complessi risultanti sono utili come agenti di contrasto per risonanza magnetica (MRI). Molti ligandi disponibili in commercio sono particolarmente utili perché contengono gruppi funzionali che consentono veloci, elevata purezza e ad alto rendimento coniugazione a macromolecole e biomolecole attraverso esteri attivati ​​ammina-reattiva e gruppi isotiocianato o tiolo-reattiva maleimides. Mentre metalation di questi ligandi è considerato conoscenza comune nel campo della chimica bioconjugation, sottili differenze nelle procedure metalation devono essere presi in considerazione nella scelta dei materiali metallici di partenza. Inoltre, più opzioni per la purificazione e la caratterizzazione esistono, e la scelta della procedura più efficace dipende in parte dalla selezione delle materie prime. Queste sottili differenze sono spesso trascurati nei protocolli pubblicati. Qui, il nostro obiettivo è quello di dimostrare metodi comuni per metalation, purificazione e caratterizzazione di complessi di lantanidi, che possono essere usati come agenti di contrasto per risonanza magnetica (Figura 1). Ci aspettiamo che questa pubblicazione permetteranno agli scienziati biomedici di incorporare le reazioni di complessazione lantanidi nel loro repertorio di reazioni comunemente usate dai facilitando la selezione delle materie prime e metodi di purificazione.

Watch this Scientific Journal Video about Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging at JoVE.com

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Dimostriamo la metalation, purificazione e caratterizzazione di complessi di lantanidi. I complessi qui descritto può essere coniugato con macromolecole di attivare il rilevamento di queste molecole usando la risonanza magnetica.

Preparazione, purificazione e caratterizzazione di complessi lantanidi per l&#39;uso di agenti di contrasto per Risonanza Magnetica

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

preparation-purification-characterization-lanthanide-complexes-for

Research

JoVE Journal

Biology

14.9K Views.  Wayne State University. Dimostriamo la metalation, purificazione e caratterizzazione di complessi di lantanidi. I complessi qui descritto può essere coniugato con macromolecole di attivare il rilevamento di queste molecole usando la risonanza magnetica.

Video: Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la RM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Ricerca

Biologia

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Etichettatura in vitro di cellule staminali embrionali umane per la Risonanza Magnetica

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Contrasto di fase e interferenza differenziale contrasto (DIC) Microscopia

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolamento e purificazione di Drosophila Neuroni periferici da Magnetic Ordinamento Bead

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Microiniezione di embrioni Medaka per essere utilizzato come organismo modello genetico

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Automated interazione idrofobica colonna di selezione cromatografia per l&#39;utilizzo in purificazione di proteine

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

Quantificare miscelazione con risonanza magnetica

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Generazione e Purificazione di umani INO80 rimodellamento della cromatina Complessi e Sottocomplessi

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

Preparazione, purificazione e caratterizzazione di complessi lantanidi per l'uso di agenti di contrasto per Risonanza Magnetica

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