这项工作得到了美国国立卫生研究院NHLBI R35HL155657（N.M.）和John C. Parker教授（N.M.）的支持。我们要感谢塞拉·阿奇博尔德女士的有益评论

该研究得到了北卡罗来纳大学教堂山分校机构审查委员会的批准（协议编号：14-2108）。
1. 从献血者那里采集血液
<ol><li>使用 21 G 针头将干净的静脉穿刺收集到肘前静脉中。丢弃前 3 mL 血液，因为这部分血液可能含有来自血管周围细胞的 TF。</li>
	<li>将 2.7 或 1.8 mL（取决于试管的大小）血液抽入含有 3.2% 柠檬酸钠 （0.109 mol/L） 的真空吸液器中。不要过度填充或不足管子。采血后立即轻轻倒置试管以分散柠檬酸钠。</li>
	<li>如果试管在加工前运输，请避免搅拌。在采血后2小时内制备血浆。</li>
</ol>2.阴性对照血浆的制备
注意：在最终冷冻之前的任何时候都不应将样品放在冰上。
<ol><li>使用来自健康志愿者的全血制备阴性对照血浆。采血后立即制备无血小板血浆，如下所示。<ol><li>将血液样本从真空管转移到 15 mL 管中。</li>
		<li>在室温（20-24°C）下以2,500× g 离心血液样品15分钟，无需制动器。</li>
		<li>将上清液（贫血小板血浆）转移到新管中，并在相同条件下重复旋转。</li>
		<li>将上清液（血小板耗竭血浆）转移到新管中。</li>
		<li>将样品分装成 ≥100 μL 等分试样。在试管底部留下约 100 μL。</li>
		<li>立即将血小板耗尽的血浆冷冻在-80°C（图1）。</li>
	</ol></li>
</ol>3.阳性对照血浆的制备
注意：对脂多糖的反应因人而异。
<ol><li>将血液样本从真空吸尘器转移到 15 mL 试管中。</li>
	<li>使用来自 大肠杆菌 O111：B4（10μg/ mL）的脂多糖（LPS）刺激的健康志愿者的全血制备阳性对照血浆，在37°C下搅拌5小时。</li>
	<li>孵育5小时后，按照步骤2.1.2至2.1.6进行操作。</li>
</ol>4. 从血浆中分离细胞外囊泡
注意：EV 颗粒可能不可见。解冻时间取决于样品体积，但我们通常在37°C下解冻100μL血浆30分钟。
<ol><li>在37°C下解冻等离子体样品。</li>
	<li>在 1.5 mL 试管中向每 100 μL 血浆样品中加入 1 mL 不含钙的 HBSA [HBSA-Ca（-）] 缓冲液 [137 mM NaCl、5.38 mM KCl、5.55 mM 葡萄糖、10 mM HEPES、0.1% （w/v） 牛血清白蛋白]。</li>
	<li>在4°C下以20,000× g 离心血浆样品15分钟。</li>
	<li>将上清液吸至20μL，而不会干扰EV沉淀。</li>
	<li>通过向每个试管中加入1mL HBSA-Ca（-）缓冲液来重新配制EV沉淀，在EV沉淀的位置上下移液，并在4°C下以20,000× g 第二次离心15分钟之前涡旋每个试管。</li>
	<li>将上清液吸至20μL，而不会干扰EV沉淀。</li>
	<li>在80μLHBSA-Ca（-）中重新配制EV沉淀，并在EV沉淀的位置上下移液（图2）。</li>
</ol>5. 选项：使用超速离心机分离小的细胞外囊泡
<ol><li>在步骤4.3之后，收集上清液并在4°C下以100,000× g 超速离心70分钟。</li>
	<li>将上清液吸至20μL，而不会干扰EV沉淀。</li>
	<li>通过向每个试管中加入1mL HBSA-Ca（-）缓冲液并涡旋每个试管来重新配制EV沉淀，然后在4°C下以100,000× g 进行第二次超速离心70分钟。</li>
	<li>将上清液吸至20μL，而不会干扰EV沉淀。</li>
	<li>在 80 μL HBSA-Ca（-） 中复溶 EV 沉淀。</li>
</ol>6. 细胞外囊泡组织因子活性的测量
注意：在添加到孔中之前，应将 EV 样品移液并涡旋充分混合。EDTA-二钠会抑制FXa裂解显色底物的能力。因此，在步骤6.7中，EDTA-二钠不能用作EDTA-四钠的替代品。
<ol><li>将 40 μL EV 样品加入 96 孔板的两个孔中。</li>
	<li>将 11 μL 抑制性小鼠抗人 TF IgG [（36.4 μg/mL，终浓度 7.8 μg/mL）] 加入到一个 EV 样品孔中，向另一个孔中加入 11 μL 对照小鼠 IgG [（36.4 μg/mL，终浓度 7.8 μg/mL）]。</li>
	<li>将96孔板在室温下孵育15分钟。</li>
	<li>在孵育期间，使用可靠的重组TF制备50μL/孔TF标准品（0,0.32,0.63,1.25,2.5,5,10和20pg / mL）。</li>
	<li>通过将 4 mL HBSA 与钙 [HBSA-Ca（+）] [HBSA-Ca（-） + 10 mM CaCl2，终浓度 10 mM]、800 μL 900 nM FX 的 HBSA-Ca（+）（终浓度：146.4 nM）和 120 μL 的 200 nM FVIIa 的 HBSA-CA（+）（终浓度：4.8 nM）混合来制备因子混合物。</li>
	<li>向每个孔中加入50μL因子混合溶液，用薄膜覆盖板，并在37°C的培养箱中孵育2小时。</li>
	<li>2小时后，通过加入25μLHBSA乙二胺四乙酸（EDTA）缓冲液[HBSA-Ca（-）+ 25mM EDTA-四钠，终浓度5mM]并在室温下孵育5分钟来停止FXa生成。</li>
	<li>将FXa裂解的显色底物复溶至4mM（向25mg小瓶中加入8.7mL蒸馏水）。</li>
	<li>向每个孔中加入25μL显色底物溶液，用薄膜覆盖板，然后用铝箔覆盖以避光，并在37°C下孵育15分钟。</li>
	<li>孵育15分钟后，用针头或以1,500× g 离心1分钟除去气泡。</li>
	<li>在读取之前，使用酶标仪读取 405 nm 的板，并进行一次混合。</li>
	<li>使用使用可靠的重组 TF 生成的标准曲线将每个孔的 FXa 生成转换为 TF 活性。</li>
	<li>使用公式 （1） 计算 TF 依赖性 FXa 生成（EVTF 活性）： TF依赖性FXa生成（EVTF活性[pg/mL]）=总FXa生成（对照IgG孔）-TF非依赖性FXa生成（抗TF IgG孔）（图3）（1）</li>
</ol>

提高细胞外囊泡中组织因子活性测定的灵敏度

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+factor-bearing+exosome+secretion+from+human+mechanically+stimulated+bronchial+epithelial+cells+in+vitro+and+in+vivo.">Tissue factor-bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo.</a> Journal of Allergy and Clinical Immunology. 130 (6), 1375-1383 (2012).</li></ol>

没有需要披露的竞争利益。

在这里，介绍了我们内部EVTF活性测定的方案。该协议有三个关键步骤。复溶 EV 沉淀时，即使在 EV 沉淀不可见的位置，也要在 EV 沉淀的位置上下移液。EV 沉淀的不完全复溶将导致样品的 EVTF 活性值出现假阴性或低估。其次，在方案步骤6.5中使用HBSA-Ca（+）至关重要，因为没有钙就无法产生FXa生成。第三，使用EDTA-四钠而不是EDTA-二钠来阻止FXa的产生至关重要，因为后者会抑制FXa裂解显色底物的能力...

凝血是通过因子 （F） VII/VIIa 与组织因子 （TF）1 的结合开始的。TF/FVIIa 复合物可激活 FIX 和 FX 以激活凝血1。全长膜结合TF有两种形式：加密和主动。此外，还有一种可变剪接形式的TF（asTF）。细胞膜外叶中的鞘磷脂和磷脂酰胆碱将 TF 维持在加密状态 2,3,4。当细胞被激活或损伤时，磷脂混血酶将磷脂酰丝氨酸和其他带负电荷的磷脂转移到外叶1。细胞的活化还导致酸性鞘磷脂酶易位到外叶，在那里它将鞘磷脂降解为神经酰胺5。这两种机制将加密的 TF 转换为活动形式。也有人提出，蛋白质二硫键异构酶介导加密TF中Cys186和Cys209之间的二硫键形成，从而导致TF 6,7,8的解密。asTF也存在于循环中，但缺乏跨膜结构域，因此可溶性9,10。重要的是，与全长活性TF10,11相比，asTF具有非常低的促凝血活性水平。
细胞外囊泡 （EV） 从静息、活化和垂死的宿主细胞以及癌细胞中释放12。EV 表达来自其亲本细胞的蛋白质12。活性 TF 携带 EV 从活化的单核细胞、内皮细胞和肿瘤细胞释放到循环中 13,14,15。血浆中TF的水平可以通过基于活性和抗原的测定来测量。基于抗原的检测包括 ELISA 和流式细胞术16。有两种不同的基于活性的测定：一期和两阶段 TF 活性测定。一阶段测定基于基于血浆的凝血测定。将含TF的样品加入血浆中，并在再钙化后测量形成凝块的时间。两阶段测定通过添加 FVII 或 FVIIa、FX 和钙来测量样品的 FXa 生成。FXa水平使用被FXa切割的底物确定。
在一阶段和两阶段TF活性测定中，TF浓度均使用重组TF生成的标准曲线测定。两阶段检测比一阶段检测具有更高的灵敏度和特异性。许多研究已经证实，基于活性的检测比基于抗原的检测具有更高的灵敏度和特异性17,18,19,20,21。此外，我们的内部活性检测比商业活性检测具有更高的灵敏度和特异性22.健康个体在血浆中的EVTF活性水平非常低或检测不到。相比之下，患有癌症、肝硬化、脓毒症和病毒感染等病理疾病的个体具有可检测到的 EVTF 活性水平，这与血栓形成、弥散性血管内凝血、疾病严重程度和死亡率相关 23,24,25,26,27,28。在这里，我们将介绍这种内部两阶段 EVTF 活性测定。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL tube for 20,000 x g centrifuge</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Fisher Scientific (Catalog number: 05-408-129).</td>
		</tr>
		<tr>
			<td>1.5 mL tube for ultracentrifuge</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Beckman Coulter (Catalog number: 357448)</td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from VWR (Catalog number: 89039-666)</td>
		</tr>
		<tr>
			<td>21 G x .75 in. BD Vacutainer Safety-Lok Blood Collection Set with 12 in. tubing and luer adapter</td>
			<td>BD</td>
			<td>367281</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the one from Globe Scientific (Catalog number: 120338).</td>
		</tr>
		<tr>
			<td>BD Vacutainer Citrate Tubes</td>
			<td>BD</td>
			<td>363083</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 1.5 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the Centrifuge 5417R (Eppendorf).</td>
		</tr>
		<tr>
			<td>Centrifuge for 15 mL tube</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the Centrifuge 5810R (Eppendorf).</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid tetrasodium salt dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>E6511</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FVIIa</td>
			<td>Enzyme Research Laboratory</td>
			<td>HFVIIa</td>
			<td>The solution should be diluted with HBSA-Ca(+).</td>
		</tr>
		<tr>
			<td>Human FX</td>
			<td>Enzyme Research Laboratory</td>
			<td>HFX1010</td>
			<td>The solution should be diluted with HBSA-Ca(+).</td>
		</tr>
		<tr>
			<td>Inhibitory mouse anti-human tissue factor IgG, clone HTF-1</td>
			<td>Fisher Scientific</td>
			<td>550252</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Escherichia coli O111:B4</td>
			<td>Sigma Aldrich</td>
			<td>L2630</td>
			<td>There are several lipopolysaccharide from different E. coli. Different lipopolysaccharide have different potential to activate monocytes.</td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Sigma Aldrich</td>
			<td>I5381</td>
			<td></td>
		</tr>
		<tr>
			<td>Pefachrome FXa 8595</td>
			<td>Enzyme Research Laboratory</td>
			<td>085-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>any company</td>
			<td>N/A</td>
			<td>We use the SpectraMax i3x from Molecular Devices</td>
		</tr>
		<tr>
			<td>Re-lipidated recombinant tissue factor, Dade Innovin</td>
			<td>Siemens</td>
			<td>10873566</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima TLX</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman Coulter</td>
			<td>TLA-55</td>
			<td></td>
		</tr>
	</tbody>
</table>

extracellular vesicle tissue factor activity assay

组织因子 （TF） 是因子 （F） VII 和 FVIIa 的跨膜受体。TF/FVIIa 复合物通过激活 FIX 和 FX 启动凝血级联反应。TF 以细胞外囊泡 （EV） 的形式从细胞释放到循环中。TF 阳性 （+） EV 水平在各种疾病中升高，包括癌症、细菌和病毒感染以及肝硬化，并且与血栓形成、弥散性血管内凝血、疾病严重程度和死亡率有关。有两种方法可以测量血浆中的 TF+ EV：基于抗原和活性的测定。数据表明，基于活性的检测比基于抗原的检测具有更高的灵敏度和特异性。本文介绍了我们基于两阶段 FXa 生成测定的内部 EVTF 活性测定。将FVIIa，FX和钙添加到含TF + EV的样品中，以在存在和不存在抗TF抗体的情况下产生FXa，以区分TF依赖性FXa世代和TF非依赖性FXa世代。使用 FXa 切割的显色底物来测定 FXa 水平，而使用可靠的重组 TF 生成的标准曲线用于测定 TF 浓度。这种内部 EVTF 活性测定比商业 TF 活性测定具有更高的灵敏度和特异性。

成功的结果给出的阳性对照值为 ≥0.5 pg/mL，阴性对照值为 <0.5 pg/mL。对于阳性对照，最好找到 EVTF 活性为 >1.0 pg/mL 的高 LPS 反应者。代表性结果显示了从 11 名健康供体的全血血浆中分离出的 EV 的 EVTF 活性，有和没有 LPS 激活（图 4）。11 个供体中有 6 个（捐献者 2、4、5、8、10、11）是中高 LPS 反应者，而 11 个捐献者中有 5 个（捐献者 1、3、6、7、9）是低 LPS 反应者。
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Watch this Scientific Journal Video about High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis at JoVE.com

作者聚焦：用于血浆诊断的高灵敏度组织因子活性检测 

在这里，我们描述了一种内部细胞外囊泡组织因子活性测定。基于活性的测定和基于抗原的测定已被用于测量人血浆样品中细胞外囊泡中的组织因子。与基于抗原的检测相比，基于活性的检测具有更高的灵敏度和特异性。

细胞外囊泡组织因子活性测定

Tissue factor (TF) is a transmembrane receptor for factor (F) VII and FVIIa. The TF/FVIIa complex initiates the coagulation cascade by activating both FIX ...

不同疾病患者的血浆中存在低水平的组织因子阳性细胞外囊泡。然而，大多数基于抗原的检测方法，包括 ELISA 和流式细胞术，不具备检测这些低水平的灵敏度。许多论文采用ELISA或流式细胞术来检测血浆中的组织因子。我们的方案以高灵敏度和特异性测量血浆中的组织因子活性，尽管由于使用功能测定而存在一些变异性。有商业检测试剂盒可以测量血浆中组织因子活性的水平。然而，这些试剂盒不含抗组织因子抗体，因此无法区分组织因子依赖性和组织因子非依赖性因子 X 生成。

extracellular-vesicle-tissue-factor-activity-assay

Research

JoVE Journal

Medicine

Extracellular Vesicle Tissue Factor Activity Assay

656 次观看.  University of North Carolina at Chapel Hill. 不同疾病患者的血浆中存在低水平的组织因子阳性细胞外囊泡。然而，大多数基于抗原的检测方法，包括 ELISA 和流式细胞术，不具备检测这些低水平的灵敏度。许多论文采用ELISA或流式细胞术来检测血浆中的组织因子。我们的方案以高灵敏度和特异性测量血浆中的组织因子活性，尽管由于使用功能测定而存在一些变异性。有商业检测试剂盒可以测量血浆中组织因子活性?...

视频: 作者聚焦：用于血浆诊断的高灵敏度组织因子活性检测 

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

实验转移的含量

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

科研

医学

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

胆固醇外流检测

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

下腔静脉间置移植物在小鼠模型中植入

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;:单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.

In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system.

血管性血友病因子-血小板串形成的流室模型研究血管血友病因子的病理生理学

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

危重病人全血内毒素血症检测的内毒素活性测定

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

小鼠肌功能的无创评价

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...