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 border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:212px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:216px;">Company</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">pFc-N1 plasmid</td>
			<td style="width:203px;">To be submitted</td>
			<td style="width:216px;">Addgene</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">HEK293 cells</td>
			<td style="width:203px;">CRL-1573</td>
			<td style="width:216px;">American Type Culture Collection</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">DMEM</td>
			<td style="width:203px;">10-013-CV</td>
			<td style="width:216px;">Mediatech - Corning</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">F2442</td>
			<td style="width:216px;">Sigma</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">100X Pen-Strep (10,000 U/ml)</td>
			<td style="width:203px;">15140-122</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">0.05% Trypsin-EDTA</td>
			<td style="width:203px;">25300054</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Lipofectamine 2000</td>
			<td style="width:203px;">11668030</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Dynabeads &#8211; Protein G</td>
			<td style="width:203px;">10003D</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Bovine Serum Albumin</td>
			<td style="width:203px;">A3294</td>
			<td style="width:216px;">Sigma</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Depression slides</td>
			<td style="width:203px;">71878-06</td>
			<td style="width:216px;">Electron Microscopy Sciences</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:212px;">Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane</td>
			<td rowspan="2" style="width:203px;">UFC901024</td>
			<td rowspan="2" style="width:216px;">Millipore</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">0.45mm syringe filter</td>
			<td style="width:203px;">83.1826</td>
			<td style="width:216px;">Sarstedt</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Dynamag-2 Magnet</td>
			<td>12321D</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:212px;">Fiji (ImageJ) Image Analysis Software</td>
			<td style="width:203px;"></td>
			<td style="width:216px;">http://fiji.sc/Fiji</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Table of Buffers/Solutions</td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Growth Media</td>
			<td style="width:203px;">DMEM + 10% FBS + Pen-Strep</td>
			<td style="width:216px;">Filter sterilize and store at 4&#176;C.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Growth Media &#8211; FBS</td>
			<td style="width:203px;">DMEM + Pen-Strep</td>
			<td style="width:216px;">Filter sterilize and store at 4&#176;C.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:212px;">Binding Buffer</td>
			<td style="width:203px;">50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2% BSA</td>
			<td style="width:216px;">Vortex until dissolved, keep on ice.</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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10.4K 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

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Monolayer의 합성, 조립, 그리고 특성화는 단백질 Monolayer의 전기에 대한 골드 Nanoparticle 필름을 보호

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

연구

생체공학

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 ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

매트릭스 수정에 의해 유도 된 세포 접착 및 탈 부착의 실시간 변화를 측정 셀 기판 임피던스와 라이브 세포 이미징을 사용하여

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

적혈구 집계 동적 조사를위한 미세 유체 제어 환경

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

향상된 세포 접착 폴리 테트라 플루오로 에틸렌에 젖은 화학 및 펩티드 고정화

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

연골 재생을위한 3D 자기 줄기 세포 집계 및 생물 반응기 성숙

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

매우 작은 공간에 팁 성장 하는 식물 세포의 신장 기능 연구를 미세 장치 개발

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

나노 제형에도 Limulus Amoebocyte Lysate (LAL) 분석 실험을 사용 하 여

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

장기간 무세포 유전자 발현의 전도를 위한 다층 미세유체 플랫폼

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 &#181;m in length with inner hydrophobic hollow channels and outer hydrophilic surfaces. JBNTs are positively charged and FNs are negatively charged. Therefore, when injected into a neutral aqueous solution, they are bonded together via noncovalent bonding to form the NM bundles. The self-assembly process is completed within a few seconds without any chemical initiators, heat source, or UV light. When the pH of the NM solution is lower than the isoelectric point of FNs (pI 5.5-6.0), the NM bundles will self-release due to the presence of positively charged FN.
NM is known to mimic the extracellular matrix (ECM) morphologically and hence, can be used as an injectable scaffold, which provides an excellent platform to enhance hMSC adhesion. Cell density analysis and fluorescence imaging experiments indicated that the NMs significantly increased the anchorage of hMSCs compared to the negative control.

The goal of this protocol is to show the assembly of a biomimetic nanomatrix (NM) with Janus base nanotubes (JBNTs) and fibronectin (FN). When co-cultured with human mesenchymal stem cells (hMSCs), the NMs exhibit excellent bioactivity in encouraging hMSCs adhesion.

야누스 베이스 나노튜브와 줄기 세포 접착을 위한 섬유네틴을 갖춘 바이오미메틱 나노 매트릭스의 제조

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...