Name: high precision zinc isotopic measurements applied to mouse organs
Uploaded: 2015-05-22T13:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about High Precision Zinc Isotopic Measurements Applied to Mouse Organs at JoVE.com

FM acknowledges funding from the ANR through a chaire d&#8217;Excellence IDEX Sorbonne Paris Cit&#233;, the INSU through a PNP grant, the Institut Universitaire de France as well as the Labex UniverEarth program at Sorbonne Paris Cit&#233; (ANR-10-LABX-0023 and ANR-11-IDEX-0005-02). We also thank funding from the European Research Council under the European Community&#39;s H2020 framework program/ERC grant agreement #637503 (Pristine).

NOTE: Procedures involving animals have been approved by the Institutional Animal Care and Use Committee (IACUC) at the Universit&#233; Paris Diderot.
1. Preparation of Materials
<ol>
	<li>Sub-boil distill 1 L of the acids (HNO3, HBr) in order to purify them from impurity.</li>
	<li>Clean the beakers and tip adaptor in a hot (~ 100 &#176;C) concentrated HNO3 acid bath for at least two days.</li>
	<li>Wash the pipette tips in a cold 3 N HNO3 bath for several days and rinse individually three times with de-ionized water.</li>
</ol>
2. Sample Preparation
<ol>
	<li>Anesthetize the mice by intraperitoneal injection of ketamine and xylazine. Assess anesthesia by the toe pinch method.</li>
	<li>Collect the blood by a cardiac puncture in the presence of heparin in 1.5 ml tubes.</li>
	<li>Separate the plasma from blood cells by centrifugation (10 min, 1,500 x g) and transfer the plasma to polypropylene cryogenic vials using polypropylene tips.</li>
	<li>Remove the remaining blood from organs by cutting the hepatic vein and injecting DPBS through the heart. Assess death of the mouse by cervical dislocation.</li>
	<li>Harvest the organs with sterile stainless steel instruments, free them of surrounding fat if any, and snap-freeze them in polypropylene cryogenic vials.</li>
</ol>
3. Chemical Purification
<ol>
	<li>First, dissolve the samples in a mix of ~ 1 ml of concentrated (30%) H2O2 and ~ 1 ml of concentrated (~ 15 M) HNO3. Do all these steps inside a fume hood.
	<ol>
		<li>Place the whole organ of interest into a 15 ml Teflon beaker. Then, add the H2O2/HNO3 to the beaker5. Keep the beaker open for a few minutes in order to avoid splashes due to the reaction of oxidation of the organic matter and the release of CO2.</li>
		<li>Finally, put the beaker on a hot plate at about 100 &#176;C for a couple of hours or until the solution is perfectly clear.</li>
	</ol>
	</li>
	<li>Open the beaker and dry the solution on a hot plate at about 100 &#176;C.</li>
	<li>Once the sample is dry, add 1 ml of 1.5 N HBr to the samples; close the beaker and let it dissolve on a hot plate at 100 &#176;C for a couple of hours.</li>
	<li>Meanwhile prepare the 500 &#181;l columns.
	<ol>
		<li>Add 500 &#181;l of the AG1X8 200-400 mesh resin to the column and put it on the column rack with a trash beaker below it. Wash the resin by alternating: 5 ml of 18.2 M&#937; &#8901; cm water, 5 ml of 0.5 N HNO3, 5 ml of water, 5 ml of 0.5 N HNO3, and then 5 ml of water. Condition the resin with 5 ml of 1.5 N HBr.</li>
	</ol>
	</li>
	<li>Remove the beakers from the hot plate and put them in an ultrasonic bath for about 30 min, and then let the beakers cool down to RT.</li>
	<li>Once the beaker is cooled and the resin is washed, open the beaker. Put the tip adaptor to the syringe, add a pipette tip; pipette the 1 ml of sample and load it onto the resin (very slowly in order not to agitate the resin).</li>
	<li>Once all the liquid passes through the column, add 5 ml of 1.5 N HBr.</li>
	<li>Once the 5 ml of 1.5 N HBr pass through the column, replace the trash beaker with a clean 15 ml beaker.</li>
	<li>Add 5 ml of 0.5 N HNO3 2.5 ml at a time. At this stage the Zn is eluted from the resin.</li>
	<li>Once 5 ml of HNO3 passes through the column, remove the beaker and place it on a hot plate at 100 &#176;C until dried.</li>
	<li>Remove the column from the column holder; trash the resin (use a new resin for each sample).</li>
	<li>Once the sample is dry, repeat the protocol with the same volume of acids on a smaller column (100 &#181;l) and then place it on a hot plate until dried. The sample is now ready for mass-spectrometry.</li>
</ol>
4. Mass-spectrometry Measurement
<ol>
	<li>Analyze the Zn isotopic composition on a multi collector-inductively coupled plasma-mass spectrometer (MC-ICP-MS).
	<ol>
		<li>Use the machine parameters summarized in Table 1.</li>
	</ol>
	</li>
	<li>Position the Faraday cups to collect at mass (m/z) of 62Ni, 63Cu, 64Zn, 65Cu, 66Zn, 67Zn and 68Zn.</li>
	<li>Prepare a solution containing 500 ppb Zn in 0.1 M HNO3 for isotopic analysis.</li>
	<li>Analyse the 500 ppb solution of Zn by using a spray chamber combined with a 100 &#181;l/min teflon nebulizer. For each sample, measure 30 scans (1 block of 30 cycles) in which the integration time of each scan is 8.389 sec.</li>
	<li>Correct the background by subtracting the on-peak zero intensities from a blank solution (the 0.1 M HNO3 solution used to re-dissolve the samples).</li>
	<li>Control and correct possible 64Ni isobaric interference by measuring the intensity of the 62Ni peak. Assume that the 64Ni/62Ni ratio is natural (0.2548), correct this value from the instrumental mass bias, and then remove the 64Ni on the mass 64 as: 
	64Znreal= 64Znmeasured - 64Ni = 64Znmeasured - (64Ni/62Ni)natural x 62Nimeasured.</li>
	<li>Correct the instrumental mass bias by bracketing each of the samples with a 500 ppb standard solution of the JMC Lyon Zn standard (or another available standard such as IRMM-3702). Perform the standard bracketing by dividing the 66Zn/64Zn ratio of the sample by the average of the 66Zn/64Zn ratio of the two standards measured before and after the sample minus 1 and multiplied by 1,000 (see Equation 1). Typical external precision on the JMC Lyon Zn standard is 0.05 permil/amu (2 standard deviation, 2 s.d.).</li>
</ol>

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The authors have nothing to disclose.

The reproducibility of the measurements is evaluated through replicated analyses of the same samples carried out during different analytical sessions. For example6, we have replicated the same terrestrial rock 7 times and we obtained the results reported in the Table 2.
As expected from the theory of isotopic fractionation10 and as measured in any solar system material so far (e.g.,&#160;meteorite11-13, plants3-5, deep-sea sediments14, animals15-17), the results follow a mass-dependent law (see Figure 1). &#948;68Zn is about twice &#948; 66Zn (Figure 1), because the mass difference between 68Zn and 64Zn is twice the difference between 66Zn and 64Zn. This shows that our measurements are free of isobaric interferences (that would drive the data out of the straight-line) and that zinc isotopes are fractionated out of the same isotopic pool.
For mouse organs, the limited amount of Zn in each organ has prevented us from performing many replicates of a single organ15. However, we can estimate a higher limit for the reproducibility by comparing data for the same tissue for different mice of the same age and same strain (for example for the bones&#160;of 16 week old mice, Table 3). This reproducibility is larger (0.04 vs 0.07 for the &#948;66Zn) than what was estimated from basaltic rocks, which is not surprising because it includes the heterogeneity of the samples as well as the isotopic variability between the different mice. It is therefore an overestimation of the reproducibility, and we believe that the precision on each individual organ would be similar to what we had determined on basaltic rocks. We can safely assume reproducibility better than 0.10 for the &#948; 66Zn (2 s.d.) which represents a precision 10 times larger than the variability reported between certain organs (see Figure 1 and reference 15).
Measuring the stable isotope composition of Zn will be used in the future as a diagnostic tool for diseases that modify the Zn balance of the body. For example, the zinc-rich plaques associated with the Alzheimer disease change the concentration of zinc in the serum and since the brain and the serum have different isotopic composition15 Zn isotopes could be used to detect early stage of the disease.
Most alternative methods to measure the Zn isotopic composition by MC-ICP-MS involve chemical purification in concentrated HCl media on larger columns than the one used here1-4. Our method based on micro-columns and diluted acids has low blanks and produces data which are two times more precise (50 ppm vs 100 ppm 2 s.d.). In addition, our method is very fast (due to the small size of the columns and the little amount of acid used) and is very well suited to analyze large amount of samples (as usually needed in clinical studies). The simplicity of the method would be well suited to be used in an automatic chemical purification system which would allow the measurements of a large numbers of samples.
One limitation of this approach is that only large bulk samples can be analyzed (the procedure uses ~ 1 &#181;g of Zn). Reducing the size of the samples is crucial when dealing with precious clinical samples. This method is also limited to bulk measurements, while for some applications in situ analyses may be needed. Future improvement on the technique should be in relation with improving in situ isotopic measurements by combining a laser-ablation system with the plasma mass-spectrometer (LA-MC-ICP-MS). This would allow the measurements of spatially small samples without prior chemical purification (which tends to contaminate the samples). In addition, in situ measurements will allow the measurement of the Zn isotopic composition on living tissues. To our knowledge there has only been one attempt to measure zinc isotope ratios using such a technique18 and the method is still not precise enough, however, measurements of high-precision isotope ratio by LA-MC-ICP-MS has been done for Fe19 and B20 and refining of the technique using modern lasers may lead to a major breakthrough.

The measurement of high-precision (better than 100 ppm/atomic mass unit) zinc stable isotope composition has only been possible for about 15 years thanks to the development of multi-collector plasma-source mass-spectrometers and has since been mostly applied in Earth and planetary sciences. The applications to the medical field are novel and have a strong potential as biomarkers for diseases that modify the metabolism of zinc (e.g., Alzheimer disease). This paper reports a method to measure with high precision the natural stable isotope ratios of zinc in various mouse organs. The same would be applicable to human samples. The method consists of the dissolution of the organs, the chemical purification of zinc from the rest of the atoms, and then the analysis of the isotope ratio on a mass-spectrometer.
The quality of Zn isotopic measurements is dependent on the quality of the chemical purification (purity of Zn, low blank compared to the amount of Zn present in the sample, high chemical yield of the procedure) and on the control of the instrumental bias. The high purity of the final Zn fraction is needed to remove both isobaric interferences and non-isobaric interference that create a matrix effect. Isobaric nuclides create direct interferences (e.g., 64Ni). Non-isobaric interferences generate the so-called &#8220;matrix&#8221; effect and alter the analytical precision of the measurements by changing the condition of the ionization compared to the pure zinc standard to which the samples are compared to1. A low blank (&#60; 10 ng) indicates that there is no contamination of the samples by external Zn that would bias the measured isotopic composition. As Zn isotopes can be fractionated during ion-exchange chromatography2, the collection of all the Zn atoms ensures that no isotopic fractionation occurs, which implies that the chemical procedure should have a full yield. Finally, the correction of the instrumental isotopic fractionation during the mass-spectrometry measurement is done via the &#8220;standard bracketing&#8221; method.
Therefore, the main difficulties to obtain precise measurements are controlling the external contamination (i.e., low blank), producing a full yield chemical purification that is clean of any other atoms or molecules, and correcting the instrumental isotopic fractionation on the mass-spectrometer. In this paper we will describe our analytical protocol to separate Zn from the mouse organs as well as the mass-spectrometry measurements.
The extraction is done using a low quantity of diluted acids (HBr/HNO3 media) on micro-columns (0.5 &#181;l and 0.1 &#181;l) of anion-exchange resin. It has a full yield and the measurements have an external reproducibility better than 50 ppm on the 66Zn/64Zn ratio. Another advantage of the method is that it is very fast. The method is therefore very well adapted to medical sciences, in which one needs to analyze a large number of samples compared to geosciences, where these analytical methods were developed.

<table><tbody><tr><td>Multi-collection inductively-coupled-plasma mass-spectromter</td><td>Thermo-Fisher</td><td></td><td></td></tr><tr><td>Anion-exchange resin AG1 X8 200-400</td><td>Bio-Rad</td><td>140-1443-MSDS</td><td></td></tr><tr><td>Teflon beakers</td><td>Savillex&amp;nbsp;</td><td>200-015-12</td><td></td></tr><tr><td>In-house-made teflon colunms made with shrinkable teflon</td><td></td><td></td><td></td></tr></tbody></table>

high precision zinc isotopic measurements applied to mouse organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

In 1.5 N HBr, the main zinc species (ZnBr3-) forms very strong complexes with the anion-exchange resin, while most other elements do not interact with the resin. Zinc is then recovered by changing the medium to diluted HNO3, changing the speciation of Zn to Zn2+ which is released from the resin6,7.
Isotope ratios are typically expressed as parts per 1,000 deviations relative to a standard:
<img alt="Equation 1" fo:content-width="2.5in" src="/files/ftp_upload/52479/52479eq1.jpg" />

with x = 66 or 68. The reference material used is the Zn &#8220;Lyon&#8221; standard JMC 3-0749 L1. The &#8220;Lyon&#8221; standard is the most broadly used reference material to normalize Zn isotope data. All the isotopic results reported are therefore relative. Using this reference the isotopic composition of the Earth for &#948;66Zn is 0.28 &#177; 0.058. Since the JMC-Lyon standard is not easily available, in the absence of this standard the alternative is to use the standard IRMM-3702 as a reference during the measurements and convert the results using reference 9 as: 66ZnJMC-Lyon = 66ZnIRMM-3702+0.29. The typical blank is &#60; 10ng.
Typical results obtained with this method are represented in the Figure 1, as a three-isotope plot (&#948;68Zn vs &#948;66Zn) for different mouse organs. Table 2 and 3 report results of replicated experiments of a typical terrestrial rock (a Hawaiian basalt) and of mouse red blood cells.
<img alt="Figure 1" src="/files/ftp_upload/52479/52479fig1highres.jpg" /> 
Figure 1. &#948;68Zn vs &#948;66Zn for different mouse organs. The typical error bar is 0.07 permil for &#948;66Zn and 0.15 for &#948;68Zn is shown on the figure. Data from reference 15.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MC-ICP-MS settings</td>
			<td>Neptune</td>
		</tr>
		<tr>
			<td>RF power (W)</td>
			<td>1,300</td>
		</tr>
		<tr>
			<td>Acceleration potential (V)</td>
			<td>10,000</td>
		</tr>
		<tr>
			<td>Gas flow rates</td>
			<td></td>
		</tr>
		<tr>
			<td>Ar coolant (l/min)</td>
			<td>18</td>
		</tr>
		<tr>
			<td>Ar auxiliary (l/min)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Ar sample (l/min)</td>
			<td>1-1.2</td>
		</tr>
		<tr>
			<td>Solution uptake rate (molution</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Analysis parameters</td>
			<td></td>
		</tr>
		<tr>
			<td>Number of blocks</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Number of measurements per block</td>
			<td>30</td>
		</tr>
		<tr>
			<td>Integration time (s)</td>
			<td>8.389</td>
		</tr>
		<tr>
			<td>Typical Zn concentration of samples and standard (ppb)</td>
			<td>500</td>
		</tr>
		<tr>
			<td>Typical transmission efficiency V/ppm</td>
			<td>25</td>
		</tr>
	</tbody>
</table>
Table 1: MC-ICP-MS settings for the Zn isotope measurements at the Institut de Physique du Globe de Paris.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Samples</td>
			<td>&#948;66Zn</td>
			<td>2se</td>
			<td>&#948; 68Zn</td>
			<td>2se</td>
			<td>na</td>
		</tr>
		<tr>
			<td>replicate 1</td>
			<td>0.34</td>
			<td>0.01</td>
			<td>0.68</td>
			<td>0.04</td>
			<td>4</td>
		</tr>
		<tr>
			<td>replicate 2</td>
			<td>0.34</td>
			<td>0.01</td>
			<td>0.68</td>
			<td>0.01</td>
			<td>3</td>
		</tr>
		<tr>
			<td>replicate 3</td>
			<td>0.34</td>
			<td>0.02</td>
			<td>0.67</td>
			<td>0.02</td>
			<td>4</td>
		</tr>
		<tr>
			<td>replicate 4</td>
			<td>0.36</td>
			<td>0.06</td>
			<td>0.7</td>
			<td>0.09</td>
			<td>4</td>
		</tr>
		<tr>
			<td>replicate 5</td>
			<td>0.31</td>
			<td>0.02</td>
			<td>0.65</td>
			<td>0.06</td>
			<td>4</td>
		</tr>
		<tr>
			<td>replicate 6</td>
			<td>0.33</td>
			<td>0.01</td>
			<td>0.68</td>
			<td>0.02</td>
			<td>3</td>
		</tr>
		<tr>
			<td>replicate 7</td>
			<td>0.32</td>
			<td>0.06</td>
			<td>0.63</td>
			<td>0.1</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Average</td>
			<td>0.33</td>
			<td>0.03</td>
			<td>0.67</td>
			<td>0.05</td>
			<td>7</td>
		</tr>
		<tr>
			<td>2SD</td>
			<td>0.04</td>
			<td></td>
			<td>0.05</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>a n=number of repeat measurement by MC-ICP-MS</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Zn isotopic composition of the Hawaii basalt K179-1R1-170.9. Each replicate represents a full chemical purification and the average of several independent mass-spectrometer measurements. Data from reference 8.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mouse number</td>
			<td>&#948;66Zn</td>
			<td>&#948; 68Zn</td>
		</tr>
		<tr>
			<td>11</td>
			<td>0.82</td>
			<td>1.6</td>
		</tr>
		<tr>
			<td>12</td>
			<td>0.79</td>
			<td>1.55</td>
		</tr>
		<tr>
			<td>13</td>
			<td>0.84</td>
			<td>1.65</td>
		</tr>
		<tr>
			<td>14</td>
			<td>0.87</td>
			<td>1.72</td>
		</tr>
		<tr>
			<td>Average</td>
			<td>0.83</td>
			<td>1.63</td>
		</tr>
		<tr>
			<td>2SD</td>
			<td>0.07</td>
			<td>0.15</td>
		</tr>
	</tbody>
</table>
Table 3: Zn isotopic composition of bones of mice. Each replicate represents a full chemical purification. Data from reference 15.

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High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

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Nanyang Technological University

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JoVE Journal

Chemistry

7.4K Views.  Université Paris Diderot. We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Video: High Precision Zinc Isotopic Measurements Applied to Mouse Organs

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Cancer Research

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

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Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Biochemistry

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Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

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Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

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The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

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Immunology and Infection

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Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

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Medicine

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Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

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We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

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Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

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Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

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