Name: extracellular vesicle tissue factor activity assay
Uploaded: 2023-12-29T12:30:00.000+00:00
Duration: 3 min 53 s
Description: Watch this Scientific Journal Video about High-Sensitivity Tissue Factor Activity Assay for Plasma Diagnosis at JoVE.com

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。
对于每种试剂，我们推荐以下储存和使用方法。我们将测定缓冲液和重组的底物储存在4°C下，并重复使用它们，直到我们用完它们。如果我们在 2-3 周内没有用完基材，我们会丢弃它，因为它会变黄。我们将重组TF，抗体，FVIIa和FX储存在-80°C下，并建议为一次检测制作等分试样，以避免冻融循环。
在初步研究中，我们将血浆样品在室温下放置9小时，在-80°C下重新冷冻过夜，并在第二天进行测定。作为对照，我们解冻了相同的血浆并立即重新冷冻。我们发现，与立即解冻并重新冷冻的样品相比，在室温下放置 9 小时的样品的 EVTF 活性降低了 16%（Hisada 和 Mackman，2017 年未发表的数据）。
除了对LPS的反应差异外，个体供体血浆中EV浓度的差异可以解释不同供体的不同EVTF活性。事实上，我们观察到 LPS 刺激的全血血浆中 EV 浓度的一些变化（1.9 ± 0.6 ×10 10 颗粒/mL，n = 6，平均值±标准差）30。
这种内部 EVTF 活性检测具有商业检测所不具备的几个功能。首先，该检测使用抗 TF 抗体来区分 TF 依赖性 FXa 代和非 TF 非依赖性 FXa 代。据称有三种商业活性测定法可以测量血浆中的 TF 活性，但没有一种使用抗 TF 抗体16。因此，这些商业检测仅测量总 FXa 生成量，而不是 TF 依赖性 FXa 生成量。其次，我们使用 2.4 nM 的 FVIIa（反应中的最终浓度）来最大限度地减少 FVIIa 产生的 TF 非依赖性 FXa。一种商业活性测定在反应中使用 12 nM 的 FVIIa，其背景很高22。事实上，在没有 TF31 的情况下，FVIIa 根据 FVIIa 浓度线性激活 FX。我们总结了比较这种内部EVTF活性测定和商业TF ELISA以及 表1中的活性测定的研究。
提出了柠檬酸血小板贫乏血浆 EVTF 活性的四种反应分类：零（0 至 <0.5 pg/mL）;弱（0.5 至 <1.0 pg/mL）、中等（1.0 至 <2.0 pg/mL）和强（≥2.0 pg/mL）23。值得注意的是，不同的抗凝剂会影响 EVTF 活性测定的结果。例如，与柠檬酸钠相比，在LPS刺激后使用肝素观察到更高水平的EVTF活性（供体A：4.6（柠檬酸盐）vs 28.6（肝素）pg/mL和供体B：3.4（柠檬酸盐）vs 26.0（肝素）pg/mL，分别为Hisada和Mackman未发表的数据2017）。相比之下，与柠檬酸钠相比，LPS刺激后使用EDTA观察到的EVTF活性水平较低（供体C：3.9（柠檬酸盐）vs1.4（EDTA）pg/mL，供体D：3.8（柠檬酸盐）vs0.4（EDTA）pg/mL，分别为Tatsumi和Mackman未发表的数据2016）。这些结果表明，当使用不同的抗凝剂时，EVTF活性数据没有可比性。
该方法存在一些限制。与临床检测相比，变异系数 （CV） 相对较高。事实上，在七项独立研究中，阳性对照的CV为24%17,29。使用离心沉淀分离 EV，不仅使用离心颗粒，还使用细胞碎片分离 EV。我们之前通过透射电子显微镜分析了富含血小板的血浆沉淀，并观察到血小板、EV 和细胞碎片32。我们使用固定角转子，没有经历过 20,000 × g 离心的摆出式转子。我们最近发现，可以用 100,000 × g 但不能用 20,000 × g 颗粒的小型 EV 在胰腺癌和 COVID-19 患者中具有 EVTF 活性30。这表明该协议不测量来自小型EV（例如外泌体）的EVTF活性。值得注意的是，外泌体是富含鞘磷脂的 EV，并且已经报道了携带 TF 的外泌体的存在30,33,34,35。我们建议使用 100,000 × g 在血浆中沉淀 EV，以更准确地确定样品中 EVTF 活性的总量。值得注意的是，它需要超速离心机和更长的分析时间，因为离心时间从 15 分钟增加到 70 分钟。
该方法可应用于任何疾病的血浆样本。EVTF 活性与患有不同疾病（包括癌症、COVID-19、细菌感染和肝硬化）的患者的疾病严重程度和生存率相关 23,26,27。

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><tbody><tr><td>1.5 mL tube for 20,000 x &lt;em&gt;g&lt;/em&gt; 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 &lt;em&gt;E. coli.&lt;/em&gt; 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&amp;nbsp;</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。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65840/65840fig01.jpg"> 图1：血小板耗竭血浆的制备。 该图是从 Hisada 和 Mackman29 修改而来的。缩写：PDP = 血小板耗竭血浆。 <a href="https://www.jove.com/files/ftp_upload/65840/65840fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65840/65840fig02v2.jpg"> 图2：从血小板耗竭血浆中分离细胞外囊泡。 该图是从 Hisada 和 Mackman29 修改而来的。缩写：PDP = 血小板耗竭血浆;EVs = 细胞外囊泡。 <a href="https://www.jove.com/files/ftp_upload/65840/65840fig02largev2.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65840/65840fig03v4.jpg"> 图3：细胞外囊泡组织因子活性的测量。 该图是从 Hisada 和 Mackman29 修改而来的。缩写：TF = 组织因子;EVs = 细胞外囊泡。 <a href="https://www.jove.com/files/ftp_upload/65840/65840fig03largev4.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65840/65840fig04.jpg"> 图 4：11 名健康供体全血血浆的 EVTF 活性，有和没有 LPS 激活。 在与阳性对照相同的条件下进行LPS激活。阳性对照来自已知的 LPS 反应者，而 11 名健康供体对 LPS 的反应在实验时尚不清楚。白条和黑条分别表示有和没有 LPS 激活。缩写：EVTF = 细胞外囊泡组织因子;LPS = 脂多糖。 <a href="https://www.jove.com/files/ftp_upload/65840/65840fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
表 1：比较这种内部 EVTF 活性测定、商业 TF ELISA 和活性测定的研究摘要。 缩写：EVTF = 细胞外囊泡组织因子;ELISA = 酶联免疫吸附测定。 <a href="https://www.jove.com/files/ftp_upload/65840/65840_Table%201%20(1).xlsx" target="_blank">请按此下载此表格。</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

Extracellular Vesicle Tissue Factor Activity Assay

684 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 ...

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

全组织细胞外囊的提取

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

癌症研究

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

测量人体血浆中的凝血因子V活性，用酶联凝固分析

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

免疫与感染

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

肝癌组织来源于小细胞外囊泡的富集方法

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

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 ...

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 ...

评估富含 EV 的血浆的血液高凝

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

作者聚焦：破译创伤性脑损伤患者的凝血障碍 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

使用 EV 激活凝血时间 （EV-ACT） 测定细胞外囊泡 （EV） 的促凝血活性

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...

细胞外囊泡组织因子活性测定

副本

摘要

探索更多视频

此视频中的章节

实验转移的含量

胆固醇外流检测

全组织细胞外囊的提取

测量人体血浆中的凝血因子V活性，用酶联凝固分析

肝癌组织来源于小细胞外囊泡的富集方法

下腔静脉间置移植物在小鼠模型中植入

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

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

使用 EV 激活凝血时间（EV-ACT）测定细胞外囊泡（EV）的促凝血活性

使用 EV 激活凝血时间（EV-ACT）测定细胞外囊泡（EV）的促凝血活性

细胞外囊泡组织因子活性测定

副本

摘要

探索更多视频

此视频中的章节

实验转移的含量

胆固醇外流检测

全组织细胞外囊的提取

测量人体血浆中的凝血因子V活性，用酶联凝固分析

肝癌组织来源于小细胞外囊泡的富集方法

下腔静脉间置移植物在小鼠模型中植入

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

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

使用 EV 激活凝血时间 （EV-ACT） 测定细胞外囊泡 （EV） 的促凝血活性

使用 EV 激活凝血时间 （EV-ACT） 测定细胞外囊泡 （EV） 的促凝血活性

使用 EV 激活凝血时间（EV-ACT）测定细胞外囊泡（EV）的促凝血活性

使用 EV 激活凝血时间（EV-ACT）测定细胞外囊泡（EV）的促凝血活性