This work was supported by an NSF/ARRA award (IOS 0920357) and an award from the NIH (5R21MH098463).

1 셀 제조 (주 -2 0) <ol><li> 분할 HEK293 세포 1 : - 80 %의 합류 (2~3일)와 0.05 % 트립신-EDTA 용액을 사용하여 5 60까지 5 % CO 2와 37 ° C에서 성장 미디어에서 부화. 각 조건의 경우, 2 × 100mm 요리에서 세포를 배양. </li></ol> 2 세포 형질 (1 일) <ol><li> 예컨대 리포 펙 타민 형질 감염과 같은 시약을 사용하여 플라스미드 인코딩 된 Fc 융합과 HEK293 세포를 형질 감염. 대등 한 형질 감염 효율을 초래할 다른 방법도 사용될 수있다. </li><li> 24 시간 동안 인큐베이터 형질 세포를 돌려줍니다. </li></ol> 3 셀 전파 (2 일) <ol><li> 37 ° C에 소 태아 혈청 (-FBS)없이 예열 성장 미디어. </li><li> 요리를 10 ㎖의 성장 미디어 -FBS으로 2 배 린스, 1 시간 동안 37 ° C 배양기로 요리를 반환합니다. </li><li> 3 세척 총, 10 ml의 성장 미디어 -FBS와 문화 요리를 한 번 더 씻어. </li><li> 인큐BATE 미디어를 수집하기 전에 FBS가없는 또 다른 48 시간 동안 37 ° C에서의 형질 HEK293 세포. </li></ol> 4 구슬 집계 (4 일) <ol><li> 배양 접시에서 미디어를 수집하려면, 세포 파편 펠릿 5 분 동안 500 XG에 50 ML 원뿔 튜브와 스핀에 요리의 각 쌍에서 미디어를 전송할 수 있습니다. </li><li> 30 ML의 주사기와 0.45 μm의 주사기 필터를 사용하여 원심 필터로 50 ML 원뿔 관에서 필터 미디어. </li><li> 농축 배양 배지의 볼륨이 약 500 μL (약 15 분) 때까지 4,000 XG, 4 ° C에서 원심 필터를 스핀. 모든 문화 매체가 추가 농축 될 때까지 반복합니다. </li><li> 자석에 얼음 각 샘플에 대한 1.5 ML microcentrifuge 관에서 버퍼를 바인딩 감기, 장소의 1 ml의 단백질 G 자석 구슬의 1.5 μl를 추가하고 버퍼를 제거합니다. 즉시 단백질 G 자석 구슬에 집중 배양 배지를 추가합니다. </li><li> 2 시간 동안 4 ° C에서 튜브를 회전합니다. </li><li> 경기 수자석에 에이스 튜브 및 용지를 제거합니다. 빨리 얼음 차가운 두 번 버퍼를 바인딩 1 ml에 구슬을 세척 한 다음 결합 완충액 300 μL에 구슬을 재현 탁. </li><li> 분할이 튜브, 각 튜브에 150 μL로 구슬을 재현 탁하고 각각 &quot;칼슘&quot;와 &quot;아니오 칼슘&quot;조건을 200 mM의 염화칼슘이 200 mM의 EDTA의 1.5 μl를 추가합니다. </li><li> 우울증에 전송 각 조건에서 100 ㎕를 잘 밀어 투과광 현미경 (그림 2)를 사용하여 현미경 사진을 수집합니다. 원하는 각 시점에서 각 실험에 대한보기의 다섯 분야에서 이미지를 수집합니다. </li></ol> (5) 데이터 분석 <ol><li> ImageJ에, 또는 비슷한 이미지 분석 소프트웨어, 파일 풀다운 메뉴에서 가져 오기 / 이미지 시퀀스 ...를 사용하여 오 영상 데이터 세트를 오픈 한 사용. 이러한 이미지 스택으로 열립니다. </li><li> 이미지 / 등록 ... 대화 상자에서 내가 변경1.0 픽셀로 설정하고 픽셀 너비와 픽셀 높이에 마법사 단위. </li><li> 이미지 풀다운 메뉴에서 조정 / 임계 값 ... 명령을 사용하여 이진하는 이미지를 변환합니다. 구슬 또는 구슬 집계에 기여하는 픽셀을 포함 할 수있는 임계 값을 설정하지만, 그 배경과 작은 입자를 제외합니다. 스택의 모든 이미지에 적용합니다. </li><li> 설정 측정 대화 상자의 영역과 스택 위치 상자를 확인합니다. </li><li> 풀다운 메뉴를 분석에 ... 입자를 분석하여 실행합니다. 이것은 그들의 크기 (면적) 및 그들에 식별 된 이미지를 포함한 식별 입자의 목록을 생성 할 것이다. </li><li> 실험 및 실험 조건에 대해이 과정을 반복합니다. </li></ol>

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The authors declare that they have no competing financial interests.

세포 접착은 다세포 생활의 필수적인 기능이며, 세포 표면 단백질의 광범위한 어레이에 의​​해 매개된다. 이들 중에서도, 접착 성 상세한 만 상대적으로 작은 비율에 대한 이해된다. 여기에, 우리는 편리한 에피토프 태그에 융합 분비 ectodomains의 homophilic 접착 능력을 조사하기위한 간단하고 빠른 프로토콜을 설명했다. 이 접근법은 중요한 장점을 갖는다. 단백질의 충분한 양이 100mm 요리의 일시 ...

세포 - 세포 부착은 다세포 생물의 발달 및 무결성에 필수적인 세포 표면 분자의 다양한 어레이에 의​​해 매개된다. 많은 사람들이 발견되고 남아 있지만이 부착 분자의 대부분은 식별 및 특성화되었다. 여러 가지 방법은 원자력 현미경과 표면력 분광법 9-12 같은 세포 정렬 분석, 세포 응집 분석법 1-8 및 생물 물리학 적 방법을 포함하여 세포 접착 분자의 특성 (캠), 조사하는데 사용된다. 심지어 세포주를 사용하여 시험 관내 시스템에서의 간략화 복잡성 어려운 추정 CAM의 점착 특성을 측정 할 수있다. 일반적으로, 분자는 비 - 접착 성 세포주에 형질 감염 될 때이 세포 응집을 유도하는 경우 CAM 여겨진다. 그러나,이 접착 활성의 직접적인 증거가없는 것이 분명하다. 예를 들어, 셀의 표면 안정성 또는 전달을 용이CAM도 증가 세포 집계 1,13 될 것입니다. 또한, CAM은 진정한 세포주 세포 표면 배달 또는 안정화에 필요한 다른 공동 인자가없는 경우 세포 응집을 매개하지 못할 수도있다. 이러한 복잡한 요인을 피하기 위해, 아이디어를 기반으로보다 직접적인 분석법 사용될 수 접착제 상호 작용은 세포 외 도메인의 본질적인 생화학 등록해야한다고. 비드가 처음 잉-2 CAM을 특성화하는 데 사용되는 동안, 이러한 분석법은 헤린 매개 12,14,15 밀착성을 조사하기 위해 확장되었다. C-cadherin의 ectodomain-FC 융합의 융합을 사용하여 Gumbiner 연구소는 여러 cadherin의 반복이 상호 작용 (14) homophilic에 기여하는 것으로 나타났다. 초파리로부터 protocadherins 1,15-18 Dscam 및 동종 형의 번호를 가지고 유사한 비드 응집 분석, E-카드 헤린 및 N-cadherin의 밀착성을 사용해도, 12,15 특징되었습니다 <em&gt; 19. 여기에 우리가 추정 homophilic 캠 (그림 1)의 분비 항원 - 태그 ectodomains의 접착 활동을 특성화 비교적 간단하고 빠른 분석을 설명합니다. 우리는 주로 cadherin의 상과 구성원의 특성을이 분석을 사용했다.

<table><tbody><tr><td>pFc-N1 plasmid</td><td>Addgene</td><td>&lt;span style=&quot;color:red;&quot;&gt;&lt;strong&gt;To be submitted&lt;/strong&gt;&lt;/span&gt;</td><td></td></tr><tr><td>HEK293 cells</td><td>American Type Culture Collection</td><td>CRL-1573</td><td></td></tr><tr><td>DMEM</td><td>Mediatech - Corning</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F2442</td><td></td></tr><tr><td>100x Pen-Strep (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668030</td><td></td></tr><tr><td>Dynabeads &amp;ndash; Protein G</td><td>Life Technologies</td><td>10003D</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3294</td><td></td></tr><tr><td>Depression slides</td><td>Electron Microscopy Sciences</td><td>71878-06</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane</td><td>Millipore</td><td>UFC901024</td><td></td></tr><tr><td>0.45 mm syringe filter</td><td>Sarstedt</td><td>83.1826</td><td></td></tr><tr><td>Dynamag-2 Magnet</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>Fiji (ImageJ) Image Analysis Software</td><td>&lt;a href=&quot;http://fiji.sc/Fiji&quot;&gt;http://fiji.sc/Fiji&lt;/a&gt;</td><td></td><td></td></tr><tr><td colspan="4">Table of Buffers/Solutions</td></tr><tr><td>Growth Media</td><td>DMEM + 10% FBS + Pen-Strep</td><td>Filter sterilize and store at 4 &amp;deg;C.</td><td></td></tr><tr><td>Growth Media &amp;ndash;FBS</td><td>DMEM + Pen-Strep</td><td>Filter sterilize and store at 4 &amp;deg;C.</td><td></td></tr><tr><td>Binding Buffer</td><td>50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2% BSA</td><td>Vortex until dissolved, keep on ice.</td><td></td></tr></tbody></table>

bead aggregation assays for the characterization of putative cell adhesion molecules

세포 - 세포 부착은 다세포 생물에 기초하고 세포 표면 단백질의 다양한 어레이에 의​​해 매개된다. 그러나, 이러한 단백질의 많은 접착제의 상호 작용이 불완전하게 이해된다. 여기서 우리는 추정 homophilic 세포 접착 분자의 접착력을 특성화하기위한 간단한 신속한 방법을 제시한다. 배양 된 HEK293 세포를 DNA 플라스미드가 세포 표면 단백질의 분비, 에피토프 태그가 ectodomain를 코딩 형질된다. 에피토프 태그에 특이 기능화 된 비드를 사용하여, 용해, 분비 된 융합 단백질은 배양 배지에서 캡처된다. 코팅 된 비드를 다음 homophilic 밀착성 테스트 분석법 정렬 비드 응집 분석법 또는 형광 비드에 직접 사용할 수있다. 경우에 따라, 돌연변이는 접착에 필요한 특정 아미노산 또는 도메인을 규명하는데 사용될 수있다. 이 분석은 안정한 세포주의 생산을 요구하지 않는, 발현 된 단백질의 소량 만이 필요하며, 수 ACCO사일에 mplished.

<html> 예를 들어 실험 된 Fc (NcadEC-FC)에 융합 N-cadherin의의 ectodomain 칼슘 의존 비드 집계를 보여줍니다 그림 2에 제시되어있다. 칼슘이없는 경우, 비드는 응집하는 경향이 거의 없거나 전혀 시간을 나타내고 (도 1a, C)와 골재 크기의 증가가 없다. 칼슘의 존재 하에서, 비드 골재 크기는 시간 (도 1B, C) ​​이상으로 증가함에 따라, NcadEC-FC 쇼 강력한 응집 코팅. 이 실...

Watch this Scientific Journal Video about Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules at JoVE.com

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

상상 속의 세포 부착 분자의 특성에 대한 비드 집계 분석 실험

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

bead-aggregation-assays-for-characterization-putative-cell-adhesion

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Cell-Cell Interactions

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10.5K 조회수.  Ohio State University. Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

비디오: Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

세포 막 확장 하 고 공부 하는 암 세포 접착 역동성에 있는 그들의 역할의 특성

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

연구

암 연구학

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

에 대한 접착 주파수 분석 현장에서</em셀 - 셀 인터페이스에서 교차 Junctional 분자의 상호 작용> 속도론 해석

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

생체공학

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

박테리아 - 호스트 상호 작용의 특성을 생체 모방 재료

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

면역학 및 감염병학

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

T 림프구 - 접착 분자 상호 작용의 연구에 대한 접착에서 전단 응력의 분석

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

이종 세포 시스템에서 단백질 응집을 통한 세포 간 상호 작용 측정

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

신경과학

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

내피 세포 접착 정량<em&gt; 체외</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

생물학

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

자석 구슬을 사용하여 기계 인장에 대한 응답으로 세포 표면 접착 리모델링 분석

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

MCF7 세포 단층층의 세포 기판 접착 영역 및 세포 모양 분포의 정량화

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

상상 속의 세포 부착 분자의 특성에 대한 비드 집계 분석 실험

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