AFM analysis was performed on instrumentation supported by the Pennsylvania Muscle Institute and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, the University of Pennsylvania. This work was supported by NIH grants HL62250 and AG047373. YHB was supported by post-doctoral fellowship from the American Heart Association.

<ol>
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The authors have nothing to disclose.

AFM 압입은 세포 및 조직의 강성 (탄성 계수)을 특성화하는데 사용될 수있다. 본 논문에서는 마우스의 하행 대동맥과 대동맥 궁을 분리하고이 동맥 영역 생체의 탄성 계수를 결정하는 자세한 단계별 프로토콜을 제공합니다. 우리는 지금 요약하고이 문서에 설명 된 방법의 기술적 인 문제 및 제한 사항에 대해 설명합니다. 몇 가지 기술적 인 문제는 고립과 자신의 작고 얇은 자연 주어진 마우스 대동맥의 분석에 발생할 수 있습니다. 세정 동맥 길이 방향으로 개방되는 경우, 치료는 탄성률 데이터 수집 내막 (내강 표면)을 방해하지 않도록주의해야한다. 최종 샘플 접시 과잉 PBS는 접시에 동맥의 적절한 접착을 방지하고,이 접착 전에 동맥을 건드리지 않고 종이 티슈를 이용하여 조심스럽게 제거해야한다. 단리 동맥 종이 티슈 WHI 스틱 수 있다는 점에 유의제작 과도한 PBS를 제거하려고하고,이 샘플이 손상 될 수 있습니다. 배양 접시에 접착 동맥 동안, 동맥 연신되지 않는 것이 매우 중요에만 접착제를 소량 동맥의 양쪽 끝 비트 별 적용된다. 강자성 들여 쓰기를 수행 할 때, 접착제가 도포 된 동맥의 끝에서 프로빙을 방지하는 것이 중요하다. 갓 절연 동맥 탄성률의 측정에 사용되기 때문에 마지막 시간은 최소화되어야 측정 수확 경과. 본 연구에서 사용 된 구형 팁은 500 nm의 반경을 가지므로, 최대 500 nm 인 압입 깊이를 정확하게 할 헤르츠 모델을 사용하여 분석 할 수있다. 이 압입 깊이 내막 (21)에있는 내피 세포의 상층과 상관된다. 사용될 수 - 동맥 샘플들의 서브 - 내피 층 역학 큰 콜로이드 프로브 (직경 20 ~ 10 ㎛)를 프로빙들여 쓰기 및 압입 깊이와 계산 된 강성의 변화를 22, 23을 판단 할 수 있습니다. 생물학적 시료는 보통 점탄성보다는 순수 탄성 때문에, (일정한 압입 깊이에 가해진 힘의 감쇠를 모니터링) 응력 완화 측정은 동맥 기계적 특성 (24, 25)의 점성 성분을 결정하기 위해 사용될 수있다. AFM 분석의 전위 한계는 조직의 작은 영역을 측정 스캔 크기이다. 혈관 세포를 포함한 대부분의 생체 시료가 아닌 지형적 및 생체 역학적으로 균질하며, 이러한 이질성은 잠재적 압입 사이트 압입 깊이 및 힘의 크기의 평활도에 따라 탄성률 artifactually 큰 변화가 발생할 수 시료에인가 표면. 전체적인 탄성 계수의 대표 평균 값을 얻기 위해, 반복 AFM-압입 A를 수행하는 것이 중요이전의 연구는 날카로운 피라미드 팁 AFM-덴트 탄성률이 높은 추정 결과 및 소프트 손상된 것을 보여준 바와 같이도 4에 도시 된 바와 같이, 동맥을 통한 t 복수의 임의의 위치가있다. 또한, 우리는 적절한 크기의 구형 팁을 사용하여 생물학적 시료 26, 27. 동맥 경화 우리 생체 AFM 측정 2 보강 평활근 세포의 분자 표지와 잘 일치 나타날지라도, 이들 동맥 샘플을 기계적으로 로딩되지 않는 것을 인식하는 것이 중요하며, 기계적 부하 전체에 중요한 역할을하는 것 같다 동맥 역학. AFM, myography (기계적 하중과 호환 생체 외 방법) (28)와 맥파 속도 (동맥 경화의 생체 측정) (29)에 의해 결정되는 동맥 경화의 비교 가능성이 arter의 가장 포괄적 인 이해를 제공합니다IAL 역학. 우리는 마우스 동맥 생체 강성을 측정하는 접촉 모드의 AFM을 설명 하였다. 이 방법은 임의로 대동맥의 복수의 영역에 압입하여 조직의 평균 강성을 추정한다. 조직의 큰 영역에서 강도의 공간 분포를 조사하기 위해, 최근 논문은 강제 볼륨 모드 AFM (30)에 사용하고있다. 이 방식은 동시에 강성도 변화의 맵과 조직의 표면 지형의 고 해상도 이미지를 획득. 조직 샘플의 강성을 측정하는 이외에, 포스 볼륨 모드 AFM은 세포 10,31 강도의 공간 분포를 모니터링하기 위해 셀에 사용될 수있다.

The biomechanical properties of arteries are a critical determinant in cardiovascular disease (CVD) and aging. Arterial stiffness, a major cholesterol independent risk factor and an indicator for the progression of CVD, increases with vascular injury, atherosclerosis, age, and diabetes1-8. Arterial wall stiffening is associated with increased dedifferentiation, migration, and proliferation of vascular smooth muscle cells9-12. In addition, increased arterial stiffness has been linked to enhanced macrophage adhesion1, endothelial permeability and leukocyte transmigration13, and vessel wall remodeling14,15. Thus, therapies that could prevent arterial stiffening in CVD or aging might complement currently available pharmacological interventions that treat CVD by reducing high blood cholesterol. 
AFM is a powerful analytical tool used for various physical and biological applications. AFM is increasingly used to obtain the high-resolution images and characterize the biomechanical properties of soft biological samples such as tissues and cells1,2,10,16,17 with a great degree of accuracy at nanoscale levels. A major advantage of AFM is the fact that it can be used with living cells.
This paper describes our method for measuring the elastic modulus of mouse arteries ex vivo using AFM. The described method shows how we 1) properly isolate mouse arteries (descending aorta and aortic arch) and 2) measure the elastic modulus of these tissues by AFM. Measurements of unloaded elastic moduli in arteries can help to elucidate changes in the extracellular matrix (ECM) that occur in response to vascular injury, CVD, and aging.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">BioScope Catalyst AFM system</td>
			<td style="width:191px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Eclipse TE 200 inverted microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Silicon nitride AFM probe</td>
			<td>Novascan Technologies</td>
			<td>PT.SI02.SN.1</td>
			<td>0.06 N/m cantilever; 1 &#181;m SiO2 particle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>See section 1.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5SF forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>See section 1.8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fine Scissors-ToughCut</td>
			<td>Fine Science Tools</td>
			<td style="width:119px;">14058-11</td>
			<td>See section 1.4 (medium sized)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas-T&#252;bingen spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15008-08</td>
			<td>See section 1.6 (small sized)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60mmTC-treated cell culture dish</td>
			<td>Corning</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Phosphate-Buffered Saline, 1X</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Krazy Glue instant all purpose liquid</td>
			<td>Krazy Glue</td>
			<td>KG58548R</td>
			<td>See section 2.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel-loading tips, 1-200 &#181;L</td>
			<td>Fisher</td>
			<td>02-707-139</td>
			<td>See section 2.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tip Tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>78092-CP</td>
			<td>See section 3.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-mm, clear wall glass bottom dishes</td>
			<td>TED PELLA</td>
			<td>14027-20</td>
			<td>See section 4.4</td>
		</tr>
	</tbody>
</table>

measuring the stiffness of ex vivo mouse aortas using atomic force microscopy

동맥 경직은 중요한 위험 인자 및 심혈관 질환 바이오 마커 및 노화의 특징이다. 원자력 현미경 (AFM)은 하드 (플라스틱, 유리, 금속 등)의 임의의 기판 위에 세포 표면에 이르기까지 다양한 재료에 대한 점탄성 기계적 성질을 특성화하기위한 다용도 분석 도구이다. 널리 세포의 강성을 측정하는 데 사용되지만 자주 대동맥의 강성을 측정하는 데 사용되었다. 본 논문에서는 무부하 마우스 동맥의 생체 탄성 계수를 측정하기 위해 접촉 모드 AFM을 사용하기위한 절차를 설명합니다. 우리는 마우스 대동맥의 분리를 위해 우리의 절차를 설명하고 AFM 분석에 대한 자세한 정보를 제공합니다. 이것은 레이저 빔의 배향, 스프링 상수 및 AFM 프로브의 편향 감도를 교정 한 힘 곡선 취득 단계가 설명되어있다. 우리는 또한 데이터 analy에 대한 자세한 프로토콜을 제공힘 곡선의 동생.

<html> 도 5a는 6 개월, 남성 C57BL / 6 마우스에서 하강 (흉부) 대동맥의 위상 콘트라스트 이미지를 보여줍니다. AFM 캔틸레버 직접 조직 위의 자리에 들여 쓰기를위한 준비.도 5b 및도 5c는 접촉 모드 AFM 들여 쓰기에 의해 얻어진 대표 힘 곡선을 보여줍니다. 도 5b 및도 5c에 도시 된 그린 선 구체 대한 헤르츠 모델을 이용하여 얻어지는 최적 곡선을 나타낸다. 본문에 설명 된도 5d에서, 하행 대동맥 대동맥 아치의 평균 강성 세 개별 쥐에서 측정 하였다. 하행 대동맥과 대동맥 궁은 ​​각각 층류 및 방해 흐름의 영역을 표현합니다. 그럼에도 불구하고, 자신의 (기계적으로 언로드) 탄성 계수는 ​​AFM의 결정에 따라 유사하다. 0 / 54630fig5.jpg &quot;/&gt; 그림 5 : 분석 포스 들여 쓰기 곡선과 하강 마우스 대동맥 생체 통해 AFM 캔틸레버를 나타내는 강성 데이터 (A) 위상차 현미경 사진.. 스케일 바, 100 μm의. (BC) (B) 하행 대동맥과 (C) 대동맥 궁 취득이 대표 강제 압입 곡선의 집합입니다. (D) 평균 마우스 하행 대동맥의 강성과 대동맥 아치로 하였다 위의 설명. 오차 막대는 평균 ± SEM을 보여; N = 3 독립적 인 생물 복제. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54630/54630fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

의 강성 측정<em&gt; 전의 VIVO</em원자 힘 현미경을 사용하여&gt; 마우스 대동맥

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

measuring-stiffness-ex-vivo-mouse-aortas-using-atomic-force

Nanyang Technological University

Research

JoVE Journal

Biology

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

10.4K 조회수.  University of Pennsylvania. We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

비디오: Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

예 VIVO 바이러스성 전달을 사용하여 헤어 대과에 GFP 타겟팅 표현

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

연구

생물학

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

광학 트랩을 사용하여 박테리아 세포의 굽힘 강성을 측정

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

기계 자극에 라이브 세포 응답 통합 광학과 원자 힘 현미경에 의해 연구

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

원자 힘 현미경을 사용하여 폐 조직의 미세 기계적 특성

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR - 기자의 구성과 microRNA를 사용하여 LightSwitch의 루시페라제 분석 시스템과 인간 microRNAs의 확인 대상이 자기편 세포의 모방

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

소설<em&gt; 생체</em배아 마우스 사랑을&gt; 문화 방법

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

단일 비리온 원자력 현미경 검사및 초분해성 형광 이미징을 위한 샘플 준비

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

인 - 더 - 필드 응용 프로그램에 대한 설계 및 앱 타머 - 금 나노 입자 기반 비색 분석법 개발

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

전 생체 내 공초점 현미경을 사용하여 림프절 구조 및 세포 국소화 시각화

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

라이브 셀 현미경을 사용하여 확산 중 셀 에지 돌출 역학 측정

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...