We gratefully acknowledge startup funds from Wayne State University (MJA), a grant from the American Foundation for Aging Research (SMV), and a Pathway to Independence Career Transition Award (R00EB007129) from the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (MJA).

1. Metalation using LnCl3 salts
<ol>
	<li>Dissolve the ligand in water to produce a solution of 30-265 mM. The ligand 2-(4-isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid 
	(p-SCN-Bn-DTPA) was used in this video at a concentration of 73 mM.</li>
	<li>Adjust the pH of the solution of ligand to between 5.5 and 7.0 by adding 1 M NH4OH. In this video, 0.2 mL of the 1 M NH4OH solution was used.</li>
	<li>Dissolve 1-2 equivalents of LnCl3 in water to produce a solution with a concentration of 5-1000 mM. In this video, EuCl3 
	and GdCl3 were used in concentrations of 111 mM. An excess of metal is often used to drive the metalation to completion and 
	consequently simplify purification.</li>
	<li>Add the solution of LnCl3 to the solution of ligand while stirring.</li>
	<li>After the addition of LnCl3, adjust the pH of the resulting reaction mixture to between 5.5 and 7.0 by adding 0.2 M NH4OH. A 
	total of 0.5 mL of the 0.2 M NH4OH solution was used in this video. If your ligand contains acid-sensitive functional groups, adjust 
	the pH multiple times during this step. CAUTION - If the solution becomes too basic, any base sensitive functional groups, like isothiocyanate, 
	will be rendered unusable for conjugation.</li>
	<li>Monitor the reaction via pH measurements. The reaction is complete when the pH remains constant.</li>
</ol>
2. Raising pH workup (not included in this video, but good for ligands without base-sensitive functional groups)
<ol>
	<li>Add concentrated NH4OH to the reaction mixture to adjust the pH to &ge;11. This step will precipitate any uncomplexed 
	metal as the insoluble hydroxide.</li>
	<li>Filter the supernatant through a 0.2 &mu;m filter. If the reaction mixture clogs the filter, centrifuging and decanting prior to 
	filtering is recommended.</li>
	<li>If dialysis will not be performed, remove solvent under reduced pressure (rotary evaporation or freeze drying is recommended).</li>
	<li>Steps 2.1-2.3 can be repeated if free lanthanide remains.</li>
</ol>
3. Dialysis workup
<ol>
	<li>Cut the dialysis tubing to an appropriate length (follow the manufacturer&#x2019;s guidelines) to hold the sample volume while leaving extra length (approximately 10% of the sample volume). In this video, a 100-500 dalton molecular weight cut-off (MWCO) membrane was used, but larger MWCO tubing can be used as appropriate if conjugation is performed prior to metalation. Also, dialysis cassettes can be used as an alternative to dialysis tubing if desired.</li>
	<li>If appropriate based on the manufacturer&#x2019;s guidelines, soak the cut dialysis tubing in water for 15 min at ambient temperature.</li>
	<li>Fill a dialysis reservoir (a 1 L beaker was used in this video) with water (dialysate). The dialysate volume should be approximately 100x that of the sample.</li>
	<li>Fold one end of the tubing twice and secure the folded portion of the tubing with a dialysis closure clamp. Wrap the end of the closure with a rubber band to ensure that it remains closed during dialysis.</li>
	<li>Filter the reaction mixture through a 0.2 &mu;m filter, and load the filtrate into 
	the open end of the tubing being careful not to tear the tubing. Be sure to leave enough head space to close the tubing.</li>
	<li>Fold the remaining open end of the tubing twice, secure with a closure, and wrap the closure with a rubber band as in step 3.4.</li>
	<li>Attach a glass vial containing air to the clamp on one end of the dialysis tubing using a rubber band. Attach a vial containing sand to the other clamp. These vials ensure that the tubing stays immersed in the dialysate.</li>
	<li>Place the full tubing in the dialysis reservoir that contains dialysate.</li>
	<li>Stir the dialysate using a magnetic stir plate at a slow speed (no vortexing) at ambient temperature.</li>
	<li>Change the dialysate 3x over the course of a day (in this video, the dialysate was changed at 2.5, 6.5, and 11.5 h), and then allow dialysis to continue overnight (for a total of 20-28 h of dialysis).</li>
	<li>Remove the dialysis tubing from the dialysate and carefully open one closure to remove the sample. Wash the dialysis tubing 3x with water and combine the washings with the sample.</li>
	<li>Remove the water under reduced pressure. Freeze drying is used in this video.</li>
</ol>
4. Assessment of the presence of free metal
<ol>
	<li>Dissolve the metal complex in acetate buffer (buffer preparation: Dissolve 1.4 mL of acetic acid in 400 mL water, adjust the pH to 5.8 with 
	1 M NH4OH, and add water to produce a total volume of 500 mL) and add the xylenol orange indicator (16 &mu;M in pH 5.8 buffer). In this 
	video, 0.3 mg of complex was dissolved in 0.3 mL of buffer and 3 mL of indicator solution was added.</li>
	<li>Detect the presence of free metal via observation of a color change of the indicator from yellow to violet.</li>
	<li>If desired, the amount of free metal can be quantified by creating a calibration curve1. Alternatively, the dye arsenazo III can 
	be used instead of xylenol orange2. If free metal remains, the sample should be further purified using dialysis, a desalting column, or high-performance liquid chromatography (HPLC) prior to characterization.</li>
</ol>
5. Determination of water-coordination number (q)
<ol>
	<li>Prepare a solution of the EuIII-containing complex (~1 mM) in H2O and another solution of the same concentration in 
	D2O. Prior to analysis, the D2O solution must be evaporated and dissolved in D2O three times to remove residual 
	H2O.</li>
	<li>Add the water solution to a clean cuvette, and place the cuvette into a spectrofluorometer.</li>
	<li>Perform excitation and emission scans to determine the maxima for each (~395 nm and ~595 nm, respectively).</li>
	<li>Perform a phosphorescence time-decay experiment using the following parameters: excitation and emission wavelengths determined from 
	step 5.3, excitation and emission slit widths (5 nm), flash count (100), initial delay (0.01 ms), maximum delay (13 ms), and delay 
	increment (0.1 ms). These conditions are appropriate for most complexes, but the maximum delay and increment values can be increased 
	or decreased for species with extremely long or extremely short decay times.</li>
	<li>Repeat step 5.4 with the D2O solution prepared in step 5.1.</li>
	<li>From the luminescence-decay data obtained in 5.4 and 5.5, plot the natural log of intensity versus time. The slope of these lines are 
	the decay rates (&tau;-1) (Figure 2). In this video, Microsoft Excel 2007 was used to generate the natural log plots from the raw data. Use the decay rates in the equation developed by Horrocks and coworkers (eq 1)3. If your ligand contains OH or NH groups coordinated to the metal, then the equation must be modified before use3.</li>
</ol>
eq 1:&nbsp;&nbsp; <img src="/files/ftp_upload/2844/2844eq1.jpg" alt="Equation 1" />
6. Relaxivity measurements
<ol>
	<li>Select the desired application mode on the relaxation time analyzer: T1 (longitudinal relaxation time) or T2 (transverse relaxation time).</li>
	<li>Prepare a series of samples that contain different concentrations of GdIII-containing complex in an aqueous solvent. In this video, water was used as the solvent and solutions of 10.0, 5.00, 2.50, 1.25, 0.625, and 0 mM were prepared. Other aqueous solvents or buffers could be used, but it is important to use the solvent as the blank. The final volume of the sample is specific to the instrument that is being used.</li>
	<li>Place a sample in the instrument and let it sit for 5 min to equilibrate to the temperature of the instrument (37 &deg;C in this video).</li>
	<li>Determine the relaxation time (in units of s) by adjusting the parameters of the software to obtain a smooth exponential curve for T1 or T2 (representative curves for T1 and T2 are shown in Figure 3).</li>
	<li>Repeat steps 6.3 and 6.4 for all samples including the blank.</li>
	<li>Calculate the inverse of the measured T1 or T2 values in units of s-1.</li>
	<li>Plot the T1-1 or T2-1 values versus GdIII concentration (in units of mM). Because of the hygroscopic nature of GdIII-containing complexes, confirm the concentration of GdIII using atomic absorption spectrophotometry or inductively coupled plasma mass spectrometry. Fit the plot with a straight line. A representative plot is shown in Figure 4.</li>
	<li>The slope of the fitted line is the relaxivity (r1 or r2 for T1 and T2, respectively) and has units of mM-1s-1.</li>
</ol>
7. Representative Results
Representative data for the steps in this protocol have been included in the Tables and Figures section. In addition to 
the water-coordination number and relaxivity characterization described in the protocol, it is important to characterize final products 
using standard chemical techniques. The identity of the compound can be obtained using mass spectrometry, and representative mass spectra 
showing the diagnostic isotope patterns for GdIII- and EuIII-containing complexes are shown in Figure 5. Furthermore, for non-GdIIIlanthanide-containing complexes, NMR spectroscopy can be used for identification of the product. To characterize the purity of the complex, HPLC or elemental analysis or both can be used.
<img src="/files/ftp_upload/2844/2844fig1.jpg" alt="Figure 1" /> Figure 1. General scheme for metalation and purification: Scheme depicting the general procedure for metalation and reasons for choosing different purification routes.
<img src="/files/ftp_upload/2844/2844fig2.jpg" alt="Figure 2" /> Figure 2. Luminescence-intensity plot: Representative plot of the natural log of intensity versus time from section 5. The slopes of the lines generated from similar curves acquired for water and D2O solutions are used with eq 1 to characterize the water-coordination number of EuIII-containing complexes.
<img src="/files/ftp_upload/2844/2844fig3.jpg" alt="Figure 3" /> Figure 3. Relaxation decay time curves: Representative data for (left) T1 and (right) T2 acquisition. Deviations from these curve shapes would produce unreliable data.
<img src="/files/ftp_upload/2844/2844fig4.jpg" alt="Figure 4" /> Figure 4. Relaxivity determination: A representative plot of 1/T1 versus the concentration of GdIII. The slope of the fitted line is relaxivity and has units of mM-1s-1.
<img src="/files/ftp_upload/2844/2844fig5.jpg" alt="Figure 5" /> Figure 5. Mass spectra: Representative mass spectra showing the diagnostic isotope patterns for (left) GdIII-containing complexes and (right) EuIII-containing complexes. The black Gaussian peaks represent the theoretical isotope distribution and the red lines are the actual data.

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Acta. 340, 44-48 (2002).</li><li>Menjoge, A. R., Kannan, R. M., Tomalia, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer-based+drug+and+imaging+conjugates:+design+considerations+for+nanomedical+applications.">Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications.</a> Drug Discovery Today. 15, 171-185 (2010).</li><li>Que, E. L., Chang, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+magnetic+resonance+imaging+contrast+agents+as+chemical+sensors+for+metals+in+biology+and+medicine.">Responsive magnetic resonance imaging contrast agents as chemical sensors for metals in biology and medicine.</a> Chem. Soc. 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No conflicts of interest declared.

Given the increasing number of publications that include lanthanide-based contrast agents4-14, it is important that care is taken in preparing, purifying, and characterizing products to ensure reproducible and comparable results. These complexes are often considered challenging to purify and characterize relative to organic molecules due to their paramagnetic nature and the sensitivity 
of any functional groups that could be used for bioconjugation. We have described common methods for the synthesis, purificat...

<table border="1">
 <tbody>
 <tr>
 <th>Reagents and Equipment</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 <td>EuCl3&bull;6H2O</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>ammonium hydroxide</td>
 <td>EMD</td>
 <td>AX1303-3</td>
 </tr>
 <tr>
 <td>Spectra/Por Biotech Cellulose Ester (CE) Dialysis Membrane - 500 D MWCO</td>
 <td>Fisher Scientific</td>
 <td>68-671-24</td>
 </tr>
 <tr>
 <td>Millipore IC Millex-LG Filter Units</td>
 <td>Fisher Scientific</td>
 <td>SLLG C13 NL</td>
 </tr>
 <tr>
 <td>xylenol orange tetrasodium salt</td>
 <td>Alfa Aesar</td>
 <td>41379</td>
 </tr>
 <tr>
 <td>acetic acid</td>
 <td>Fluka</td>
 <td>49199</td>
 </tr>
 <tr>
 <td>D2O</td>
 <td>Cambridge Isotope Laboratories, Inc.</td>
 <td>DLM-4-25</td>
 </tr>
 <tr>
 <td>water purifier</td>
 <td>ELGA</td>
 <td>Purelab Ultra</td>
 </tr>
 <tr>
 <td>high performance liquid chromatography and mass spectrometry</td>
 <td>Shimadzu</td>
 <td>LCMS-2010EV</td>
 </tr>
 <tr>
 <td>relaxation time analyzer</td>
 <td>Bruker</td>
 <td>mq60 minispec</td>
 </tr>
 <tr>
 <td>UV-vis spectrophotometer</td>
 <td>Fisher Scientific</td>
 <td>20-624-00092</td>
 </tr>
 <tr>
 <td>freeze dryer</td>
 <td>Fisher Scientific</td>
 <td>10-030-133</td>
 </tr>
 <tr>
 <td>pH meter</td>
 <td>Hanna Instruments</td>
 <td>HI 221</td>
 </tr>
 <tr>
 <td>spectrofluorometer</td>
 <td>HORIBA Jobin Yvon</td>
 <td>Fluoromax-4</td>
 </tr>
 <tr>
 <td>Molecular Weight Calculator version 6.46 by Matthew Monroe, downloaded October 17, 2009</td>
 <td><a href="http://ncrr.pnl.gov/software/">http://ncrr.pnl.gov/software/</a></td>
 <td>Molecular Weight Calculator</td>
 </tr>
 </tbody>
</table>

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

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

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

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

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.

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

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.

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.

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

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.

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.

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 ...

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.

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 ...

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.

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

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...