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

FM은 chaire 디부 우수 IDEX 소르본 파리 시테 통해 ANR에서 자금을 인정하고, INSU PNP 승인을 통해, 연구소 Universitaire 드 프랑스뿐만 아니라 소르본 파리 시테 (ANR-10-LabX에-0023과 ANR에서 LABEX UniverEarth 프로그램 -11-IDEX-0005-02). 우리는 또한 유럽 공동체의 H2020 프레임 워크 프로그램 / ERC 보조금 협정 # 637503 (원시)에서 유럽 연구위원회의 자금 지원을 주셔서 감사합니다.

참고 : 동물을 포함하는 절차는 Université 파리 디드로의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었습니다. 재료 1. 준비 <ol><li> 하위 종기 더러움을 정화하기 위해 산의 1 L (HNO 3의 HBr)를 증류. </li><li> 뜨거운 (~ 100 °의 C)에서 커와 팁 어댑터 청소 적어도 이틀 동안 HNO 3 산욕을 농축시켰다. </li><li> 며칠 동안 감기 3 N HNO 3 조에서 피펫 팁을 세척하고 탈 이온수 개별적으로 세 번 씻어. </li></ol> 2. 샘플 준비 <ol><li> 케타민과 자일 라진의 복강 내 주사하여 쥐를 마취. 발가락 핀치 방법으로 마취를 평가합니다. </li><li> 1.5 ㎖의 튜브에 헤파린의 존재하에 심장 천자에 의해 혈액을 수거. </li><li> 원심 분리 (10 분, 1,500 XG)에 의해 혈액 세포에서 플라즈마를 분리 및 폴리 프로필렌 (C)에 플라즈마를 전송폴리 프로필렌 정보를 사용하여 ryogenic 병. </li><li> 심장을 관통 DPBS를 간 정맥을 절단하고 주입하여 기관에서 남아있는 혈액을 제거합니다. 자궁 전위에 의해 마우스의 죽음을 평가합니다. </li><li> 멸균 스테인레스 스틸 악기와 장기를 수확있는 경우 지방 주변을 무료로, 폴리 프로필렌 극저온 유리 병에서 그들을 동결 스냅. </li></ol> 3. 화학 정제 <ol><li> 첫째, 집중 (30 %)의 ~ 1 ml의 H 2 O 2와 농축 (~ 15 M) HNO 3 ~ 1 ML의 혼합에 샘플을 녹인다. 흄 후드 내부의 모든 단계를 수행합니다. <ol><li> 15 ml의 테플론 비커에 관심있는 모든 기관을 놓습니다. 그런 다음, 비커 5 H 2 O 2 / HNO 3를 추가합니다. 유기물의 산화 반응 및 CO 2의 방출로 인한 스플래시를 피하기 위해 몇 분 동안 열기 비이커 유지. </li><li> 마지막으로, ABO 핫 플레이트에 넣어 비이커솔루션 시간이나 때까지 몇 UT 100 ° C는 완벽하게 알 수있다. </li></ol></li><li> 비커를 열고 약 100 ℃의 핫 플레이트에 솔루션을 건조. </li><li> 샘플이 건조되면, 샘플에 1.5 N의 HBr 1 ㎖을 추가; 비커를 닫고 몇 시간 100 ℃에서 핫 플레이트에 용해 할 수 있습니다. </li><li> 한편 500 μL 열을 준비합니다. <ol><li> 컬럼에 AG1X8 200-400 메쉬 수지의 500 μl를 추가하고 아래의 쓰레기 비커와 열 선반에 넣어. 5 ㎖ MΩ ⋅ cm, 물, 0.5 N HNO 3 5 ㎖, 물 5 ㎖, 0.5 N HNO 3 5 ㎖ 18.2을, 그리고 물을 5 ml의 : 교번하여 수지를 씻어. 1.5 N HBr을 5 ml의 수지를 조정. </li></ol></li><li> 비커 실온까지 냉각 할 수 후 열판에서 비커를 제거하고 약 30 분간 초음파 욕조에 넣어합니다. </li><li> 비이커 냉각시키고 수지를 세척 후, 비커를 연다. T로 끝 어댑터를 넣어그는 주사기 피펫 팁을 추가; 샘플의 1 ml에 피펫 (수지를 선동하지 않기 위해 매우 느리게) 수지에로드합니다. </li><li> 모든 액체가 컬럼을 통해 통과하면 1.5 N의 HBr 5 mL를 추가한다. </li><li> 1.5 N의 HBr의 5 ml의 칼럼을 통해 합격 한 후, 깨끗한 15 비이커와 쓰레기 비커를 교체합니다. </li><li> 한 번에 0.5 N HNO 3 2.5 ml의 5 mL를 추가합니다. 이 단계에서 아연이 수지로부터 용출된다. </li><li> HNO 3의 5 ml의 컬럼을 통과하면, 비커를 제거하고 건조 할 때까지 100 ℃의 핫 플레이트에 놓습니다. </li><li> 열 홀더에서 열을 제거; 수지 쓰레기 (각 샘플에 대한 새로운 수지를 사용). </li><li> 샘플이 건조되면, 작은 컬럼에 산 (100 μL)의 동일한 부피의 프로토콜을 반복 한 후 건조 할 때까지 핫 플레이트 위에 놓는다. 샘플은 질량 분석을위한 준비가되었습니다. </li></ol> 4. 대량 분석 측정 <ol><li> 아연 동위 원소 COMPO 분석멀티 콜렉터 - 유도 결합 플라즈마 질량 분석기 (MC-ICP-MS)에 sition. <ol><li> 표 1에 요약 시스템 파라미터를 사용한다. </li></ol></li><li> (62) 니켈, 구리 (63), (64) 아연, 구리 (65), (66) 아연, 아연 67, 68 아연의 질량 (M / Z)에서 수집하는 패러데이 컵을 놓습니다. </li><li> 동위 원소 분석을 위해 0.1 M HNO 3 500 PPB의 아연을 함유 한 용액을 준비합니다. </li><li> 100 μL / 분 테플론 분무기와 함께 분무 챔버를 사용하여 아연 500 ppb의 용액을 분석한다. 각 샘플에 대해, 각 주사의 통합 시간은 8.389 초 30 인 스캔 (30 사이클의 1 블록)를 측정한다. </li><li> 빈 솔루션 (샘플을 다시 용해하는 데 사용되는 0.1 M HNO 3 용액)에서의 피크 제로 강도를 뺀 배경을 수정합니다. </li><li> 제어 및 62 니켈 피크의 강도를 측정하여 정확한 수 (64) 니켈 동중 간섭.올바른, 64 니켈 / 62 니켈의 비율이 자연이라는 악기 질량 바이어스에서이 값을 (0.2548)을 가정 한 다음과 같은 질량 64 64 니켈을 제거 : 측정 (64) 아연 실제 = 64 아연 - 니켈 측정 (64) = 64 아연 - (64 니켈 / 62 니켈)는 62 X 자연은 니켈 측정. </li><li> JMC 리옹 아연 표준의 500 ppb의 표준 용액 (또는 IRMM-3702과 같은 다른 가능한 표준)으로 각각의 샘플을 다단계로 악기 대량 편견을 수정합니다. 이전과 샘플 1을 뺀 후에 측정 1,000 곱하여 두 표준 66 아연 / 64 아연 비율 평균하여 샘플의 66 아연 / 64 아연 비율 나눈 표준 브라케팅 수행 (수학 식 1 참조). JMC 리옹 아연 표준에 전형적인 외부 정밀도는 0.05 permil / AMU (2 표준 편차, 2 SD)입니다. </li></ol>

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저자가 공개하는 게 없다.

측정의 재현성이 다른 분석 세션 동안 수행 동일한 샘플 복제의 분석을 통해 평가된다. 예 6에서, 우리는 같은 지상파 록 7 번 복제 된 우리가 표 2에보고 된 결과를 얻었다. 동위 원소 분별 (10)의 이론에서 예상하고 지금까지 어떤 태양계 물질의 측정 (예, 운석 11-13, 3-5, 심해 퇴적물 (14), 동물 15-17 식물)으로, 결과는 ...

높은 정밀도의 측정은 (더 이상 100 PPM / 원자 질량 단위) 아연 안정 동위 원소 조성은 다중 콜렉터 플라즈마 소스 질량 분석기의 개발로 약 15 년 감사 가능한 왔으며 보낸 주로 어스 적용된 행성 과학. 의료 분야에 응용 프로그램은 소설이며, 아연의 대사 (예를 들어, 알츠하이머 병)를 수정 질환의 바이오 마커로서 강력한 잠재력을 가지고있다. 이 논문은 여러가지 마우스의 장기에 고정밀 아연 천연 안정 동위 원소 비율을 측정하는 방법을보고한다. 같은 인간의 샘플에 적용될 수있다. 이 방법은 장기 원자의 나머지 아연의 화학적 정제 한 다음 질량 분광기상에서 동위 원소 비의 분석 용해 이루어져있다. 아연 동위 측정 품질은 화학 정제의 품질 (아연의 순도, 낮은 빈 샘플 콘텐츠에 의존샘플에 존재하는 아연의 양에 ared, 높은 화학적 과정의 수율)과 계기 바이어스 컨트롤. 최종 Zn으로 분획의 고순도가 매트릭스 효과를 동중 간섭 비 등압 간섭 모두를 제거하는데 필요하다. 동중 핵종 직접 간섭 (예를 들어, 64 니켈)을 만듭니다. 비 동중 간섭은 소위 &quot;매트릭스&quot;효과를 생성하고, 시료 1로 비교되는 순수한 아연 표준에 비해 이온화 상태를 변경하여 측정 분석의 정밀도를 변경. 저 빈 (&lt;10 NG)는 외부 아연에 의한 샘플의 어떤 오염이 없음을 나타냅니다 바이어스 측정 된 동위 원소 조성 것. 아연 동위 원소가 이온 교환 크로마토 그래피에 2 중 분획 수있는 바와 같이, 모든 아연 원자의 컬렉션에는 동위 원소 분획은 화학적 방법으로 전체 수율을 가져야한다는 것을 의미하는 발생하지 않는 것을 보장. 마지막으로, 질량 분석기 측정시 악기 동위 원소 분별의 보정은 &quot;표준 브라케팅&quot;방법을 통해 이루어집니다. 따라서, 주요 어려움 얻기 위해 정확한 측정은 다른 원자 또는 분자의 깨끗 전체 수율 화학 정제를 생산, (빈 즉, 낮음) 외부 오염을 제어하고, 질량 분석기에 쓸모 동위 원소 분류를 수정하고 있습니다. 본 논문에서는 마우스의 장기뿐만 아니라 질량 분광법 측정으로부터 아연을 분리하기 위해 분석 프로토콜을 설명한다. 추출은 음이온 교환 수지의 마이크로 컬럼 (0.5 μL, 0.1 μL)에 희석 산의 낮은 양 (HBr로 / HNO 3 용지)를 사용하여 수행된다. 그것은 전체 수율을 가지며, 측정 재현성을 외부 Zn으로 66 / 64 아연 비율에 더는 50ppm 이하를 갖는다. 메트의 또 다른 장점OD는 아주 빠르다는 것이다. 방법은 하나 따라서 잘 이러한 분석 방법이 개발되었다 지학에 비해 많은 수의 샘플을 분석 할 필요가있는 의료 과학하도록 구성된다.

<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

우리는 마우스 기관에서 높은 정밀도 아연 동위 원소 비율로 측정 할 수있는 방법을 제시한다. 아연은 자연스럽게 마우스 기관 사이에 분획있다 (5) 안정 동위 원소 (64 아연, 아연 66, 67 아연, 68 아연 및 아연 70)로 구성되어있다. 우리는 먼저 아연 원자를 확보하기 위해 다른 기관을 용해하는 방법을 보여줍니다; 이 단계는 HNO 3, H 2 O (2)의 혼합물에 의해 실현된다. 우리는 그 다음 묽은의 HBr / HNO 3 매체에 음이온 교환 크로마토 동중 간섭 (예, 니켈)로부터 특히 다른 모든 요소에서 아연 원자를 정제. 이러한 처음 두 단계는 고순도 화학 물질을 사용하여 깨끗한 실험실에서 수행된다. 마지막으로, 동위 원소 비율은 낮은 해상도, 다중 콜렉터 유도 결합 플라스마 질량 분석계를 사용하여 측정된다. 샘플 correc 인 스프레이 챔버 및 질량 분광기에 의해 유도 된 분획을 사용하여 동위 원소 주입표준 (표준 브라켓 기술)의 비율로 샘플의 비율을 비교하여 테드. 이것은 전체 전형적인 절차는 50ppm (2 SD) 재현성 동위 원소 비를 생성한다.

1.5 N의 HBr에서 메인 아연 종 (ZnBr3-) 음이온 교환 수지와 함께 강한 착물 형성은 대부분의 다른 요소는 수지와 상호 작용하지 않는 상태. 아연은, 희석 HNO 3 매질을 변화 -6,7- 수지로부터 방출되는 Zn을 Zn과의 분화 ​​2+ 변경함으로써 회수된다. 동위 원소 비율은 전형적으로 표준 편차에 대하여 1,000 분율로 표현된다 : <img alt="식 (1)"...

Watch this Scientific Journal Video about High Precision Zinc Isotopic Measurements Applied to Mouse Organs at JoVE.com

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

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

마우스 기관에 적용 높은 정밀도 아연 동위 원소 측정

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 ...

high-precision-zinc-isotopic-measurements-applied-to-mouse-organs

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

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

비디오: 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.

포괄적 인 절차를 평가하는<em&gt; 생체</em&gt; 암 나노 의학의 성능

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

연구

암 연구학

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

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

준비 및 평가<sup&gt; 99m</sup&gt; Bioorthogonal 화학을 사용하여 사전이 타겟팅에 대한 세자리 킬레이트를 Tc를 표지

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

화학

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

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 ...

생화학

Shotgun Lipidomics of Rodent Tissues

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.

설치류 조직의 산탄총 지질체

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

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

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

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.

마우스의 여러 장기에서 Zika 바이러스의 격리 및 정량화

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

면역학 및 감염병학

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

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.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

신체 구성 및 높은 지방 연 준 쥐에서 대사 배우면 분석

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

의학

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

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.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

부드러운 근육 세포 계보 추적 마우스의 고급 동맥 경 화성 병 변에서 세포 성분의 정량 분석

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

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.

마우스에서 간세포 핵 Ploidy의 추정을 위한 situ 방법에서 높은 처리량

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

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

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

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

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

양전자 방출 단층 촬영을 사용한 실험적 자가면역 뇌척수염 마우스 모델에서 CD19+ B 세포 이미징

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

마우스 기관에 적용 높은 정밀도 아연 동위 원소 측정

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