我们感谢 Drs, 马丁. 施密特和线 Fraemohs。这项工作得到荷兰科学研究基金会 (ZonMW 大帝 016.126.358, 兰德斯塔纳输血研究基金会 (LSBR Nr 1638) 和德意志 Forschungsgemeinschaft (SFB1123/A2) 的支持, 获得 R.R.K。

这里描述的所有方法都得到了马斯特里赫特大学医学伦理和动物伦理委员会的批准。
1. 以人体细胞为基础的流动检测方法
<ol><li>
人血液中血小板的分离
	<ol>
<li>在枸橼酸 (3.2%) 抗凝血中提取静脉血液。</li>
		<li>添加1/15 酸柠檬酸葡萄糖 (80 毫米柠檬酸三钠, 52 毫米柠檬酸和183毫米葡萄糖) 的血液。</li>
		<li>离心机在 350 x g 无刹车15分钟获得富含血小板的血浆 (PRP)。</li>
		<li>将上清液转移到15毫升锥形管上, 加入1/20 体积的加性。</li>
		<li>离心机在1110克无刹车15分钟, 获得血小板不良血浆 (PPP)。</li>
		<li>放弃上清。切断末端的吸管提示 (P 1000), 使其宽孔, 这有助于预防血小板活化, 并小心地暂停血小板颗粒在同一体积的 PRP 血小板缓冲 pH 值 6.6 (PB: 136 毫米氯化钠, 10 毫米 Hepes, 2.7 毫米氯化钾, 2 毫米氯化镁2, 0.1% 牛血清白蛋白和0.1% 葡萄糖)。添加1/20 卷的加和轻轻混合。</li>
		<li>在1110克无刹车的情况下将血小板颗粒15分钟。</li>
		<li>在1毫升血小板缓冲液中去掉上清, 并小心地将血小板悬浮在上面 (pH 值 7.5)。</li>
		<li>用自动血液分析仪测定1/10 稀释液中的血小板浓度。</li>
		<li>用血小板缓冲 (pH 值为 7.5) 调整音量以达到 2 x 107/毫升的计数。</li>
	</ol>
</li>
	<li>
从人血液中分离中性粒细胞 (从 Brinkmann等) 中提取。11 )
	<ol>
<li>在肝素 (10 U/毫升) 中抽出24毫升血液作为抗凝血剂。</li>
		<li>添加6毫升的1.077 克/毫升密度聚蔗糖钠葡介质 (见材料表) 到15毫升圆锥管, 并小心地5-6 毫升全血在上面。</li>
		<li>离心机为20分钟, 在 800 x g 无刹车。</li>
		<li>吸入并丢弃透明黄顶层, 将含有粒细胞的下红色相转移到新鲜的15毫升锥形管中。通过将含有2毫米 EDTA 的 PBS 添加到最终体积为14毫升, 并在 300 x g 处无刹车的离心机中冲洗细胞。</li>
		<li>同时, 准备一个密度梯度介质 (例如, percoll, 见材料表) 工作解决方案, 混合18毫升密度梯度介质与2毫升 10x PBS 股票解决方案。用 PBS (1x) 稀释这个工作解决方案, 以获得85%、80%、75%、70% 和65% 梯度介质溶液的4.25 毫升。</li>
		<li>用密度梯度制备2锥形管, 将每一个中等百分比的2毫升分层排列在彼此之上。以最高浓度开始, 并继续递减顺序。用防水标记标记管上的层。</li>
		<li>小心层2毫升的细胞悬浮在两个准备梯度。</li>
		<li>离心机20分钟在 800 x g 不刹车在一个仔细地平衡离心机。</li>
		<li>离心后, 去除顶层和65% 层 (第一环) 与 PBMCs, 并收集到新的管白色剩余的 interphases, 直到85% 层 (第二环, 中性粒)。</li>
		<li>用 PBS 2 毫米 EDTA 填充管, 并在 300 x g 处无刹车的离心机进行10分钟冲洗。</li>
		<li>取出上清, 并暂停沉淀细胞 (通常≥95% 是中性粒蛋白) 在1毫升的汉克的缓冲 (pH 7.45), 含有10毫米 Hepes 和0.2% 人白蛋白 (化验缓冲区)。</li>
		<li>使用 hemocytometer 计数单元格, 并在 1 x 106/mL 处挂起单元格。</li>
		<li>继续荧光标记和白细胞灌注 (步骤 3.3)。</li>
	</ol>
</li>
	<li>
粘附血小板和血管细胞膜的制备
	<ol>
<li>
细胞培养皿制备 (培养细胞)
		<ol>
<li>预涂35毫米细胞培养皿与1毫升稀释的鼠尾胶原蛋白 (30 µg/毫升稀释在 PBS 过滤超过0.2 µm 过滤器)。</li>
			<li>孵育30分钟在37摄氏度, 放弃胶原蛋白溶液, 并洗一次与 PBS 的菜肴。</li>
		</ol>
</li>
		<li>
培养皿中血管细胞的培养
		<ol>
<li>分离主 (例如、HUVEC、HAoEC、HAoSMC) 或永生化血管细胞 (例如, EA。Hy926, SVEC) 生长在一个 T75 瓶与1.5 毫升细胞脱离解决方案 (例如, accutase) 汇合。添加4.5 毫升适当的生长培养基, 以中和细胞脱离溶液, 并确定细胞浓度与细胞计数室。在适当的生长培养基中悬浮 0.5 x 105细胞/mL 细胞 (例如, 完全内皮细胞生长培养基)。</li>
			<li>添加2毫升的细胞悬浮到每35毫米菜。在37°c 的湿润孵化器中培养细胞, 5% CO2直到汇合 (根据细胞类型 24–48 h)。</li>
		</ol>
</li>
		<li>
玻璃盖玻片制备 (粘附血小板)
		<ol>
<li>将玻璃盖玻片浸入盐酸-乙醇 (1.2 米/50% v/v)。</li>
			<li>用超纯水冲洗盖玻片2次。</li>
			<li>在 N2下干燥盖玻片。</li>
			<li>预涂玻璃盖玻片与100µL, 根据稀释大鼠尾胶原的灌注室通道的位置 (30 µg/毫升稀释在 PBS 过滤0.2 µm 过滤器)。</li>
			<li>孵育30分钟在 RT, 放弃胶原蛋白溶液, 并清洗盖玻片3x 与 PBS。</li>
			<li>阻断胶原蛋白涂层盖玻片与 1% BSA 在 HEPES 0.5 小时的 RT。</li>
			<li>把盖玻片洗乾净, 把它装入灌注室。分别在步骤1.1 或2.1 中概述了从人或鼠血液中分离出的血小板。</li>
		</ol>
</li>
		<li>
血小板固定化
		<ol>
<li>添加70µL 2 x 107/毫升血小板悬浮每通道, 并孵育 1.5 h 在37°c 和 5% CO2。</li>
			<li>用阻断溶液 (HEPES 中的 5% BSA) 仔细替换非黏附血小板, 在 RT 中至少30分钟. 继续荧光标记和白细胞灌注 (步骤 3.3)。</li>
		</ol>
</li>
		<li>
血管细胞单层刺激
		<ol>
<li>用含有 10 ng/毫升肿瘤坏死因子α (TNFα) 的新鲜培养基在37摄氏度和 5% CO2上替换培养基, 以刺激血管细胞。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 以小鼠细胞为基础的流动检测方法
<ol><li>
小鼠血液中血小板的分离
	<ol>
<li>使用21克针 (约1毫升) 从麻醉 (氯胺酮80毫克/千克和 medetomidine 0.3 毫克/千克) 6–8周老鼠 (3.2%) 作为抗凝剂, 用心脏穿刺抽血。</li>
		<li>添加1/6 卷的加总。</li>
		<li>离心机在 220 x g, 中制动5分钟, 获得富含血小板的血浆 (PRP)。</li>
		<li>将上清 (~ 600 µL) 加1/3 红细胞移植到新管上</li>
		<li>离心机在 95 x g 无刹车6分钟。</li>
		<li>转移上清 (PRP) 和测量血小板浓度在1/10 稀释 Tyrode 的缓冲 (TH 缓冲: 136 毫米氯化钠、5毫米 Hepes、2.7 毫米氯化钾、0.42 毫米 NaH2PO4* H2O、2毫米氯化镁2、0.1% 牛血清白蛋白和0.1%葡萄糖) 与全自动血液分析仪。</li>
		<li>通过增加1/25 容积的加 0.1 U/毫升 apyrase, 和离心机在2870克, 中制动器2分钟, 洗涤血小板。</li>
		<li>丢弃上清, 并添加600µL 的缓冲液, 1/10 卷的加成, 0.1 U/毫升 apyrase。切断吸管尖 (P 1000), 使其宽孔, 这有助于防止血小板活化, 并轻轻地暂停颗粒。</li>
		<li>离心机在2870克, 中制动器为2分钟</li>
		<li>去掉上清, 轻轻地将血小板悬浮在缓冲液中。</li>
		<li>使用 TH 缓冲区调整卷以实现 2 x 107/mL 的计数。</li>
	</ol>
</li>
	<li>
粘附血小板单分子膜的制备
	<ol>
<li>按1.3.3 制备玻璃盖玻片 (粘附血小板)。</li>
	</ol>
</li>
	<li>
血小板固定化
	<ol>
<li>添加100µL 2 x 107/毫升血小板悬浮每通道, 并孵育 1.5 h 在37°c 和 5% CO2。</li>
		<li>用阻断溶液 (HEPES 中的 5% BSA) 仔细替换非黏附血小板, 在 RT 中至少30分钟. 继续荧光标记和白细胞灌注 (步骤 3.3)</li>
	</ol>
</li>
	<li>
小鼠血小板单层的刺激作用
	<ol>
<li>在白细胞灌注前 (步骤 3.3), 在37摄氏度的 HHHSA 缓冲液中灌注凝血酶 (0.5 nM), 刺激血小板。</li>
	</ol>
</li>
</ol>3. 荧光标记和灌注白细胞 (人和 RAW264.7 或小鼠单核细胞的中性粒或 THP-1, 用于小鼠流动检测)
<ol><li>
荧光标记
	<ol>
<li>标记白细胞 (1 x 106/毫升) 与细胞 permeant 绿色荧光核酸染色 (1 µM)。在37摄氏度孵化细胞30分钟。</li>
		<li>用 PBS 以 300 x g 为5分钟的离心清洗细胞, 并将细胞悬浮到浓度为 0.5 x 10 的6细胞/mL (每个测试条件下的5毫升, 取决于流速) 在化验缓冲器中。</li>
	</ol>
</li>
	<li>
流动室化验准备
	<ol>
<li>对于细胞单层上的白细胞黏附, 从盘子中丢弃细胞培养基, 并将其组装到流动室中。将油管连接到注射器上。对于固定化血小板的白细胞黏附, 将微滑块与注射器连接。预热水浴到37°c。</li>
		<li>取一个灌注注射器 (50 毫升), 在注射器支架上安装注射器, 并在退出模式下设置泵。将泵容积设置为0毫升, 并将直径设置为26.70 毫米, 用于50毫升注射器。</li>
		<li>将弯头鲁尔连接器连接到油管的一端。将鲁尔锁耦合器放到注射器上, 并将油管与弯头鲁尔接头连接至鲁尔锁接头。将自由油管端连接到流室。</li>
	</ol>
</li>
	<li>
白细胞灌注和黏附力
	<ol>
<li>对于细胞单层上的白细胞黏附, 从盘子中丢弃细胞培养基, 并将其组装到流动室中。将油管连接到注射器上。对于固定化血小板的白细胞黏附, 将微滑块与注射器连接。</li>
		<li>对于白细胞灌注和粘附在血管-或血小板单层, 将第二管插入一个50毫升圆锥管, 其中包含检测缓冲器和填充油管与1毫升吸管, 然后挤压油管和连接到一个流动室。</li>
		<li>在白细胞灌注之前, 添加3毫米 CaCl2和2毫米氯化镁2到细胞悬浮和孵育5分钟在37°c。</li>
		<li>灌注在细胞灌注前检测缓冲液, 以去除腔室和导管中可能的空气。</li>
		<li>关闭管结束时, 挤压他们紧, 并切换油管从化验缓冲区到细胞悬浮, 防止气泡被困。灌注细胞具有适当的流速/剪切应力, 直到第一个细胞到达。</li>
		<li>灌注细胞与适当的流速/剪切应力为2分钟, 并捕获至少6在滚动和贴壁细胞之间的2–6分钟与倒置相对比/荧光显微镜 (例如, EVOS-佛罗里达州) 使用100X 放大连接数字 CCD 或 CMOS 相机。 注: 流速/剪切应力取决于灌流液的流动室尺寸和粘度。用于此处使用的灌注腔 (请参阅材料表): τ =η* 97.1 *Φ τ: 剪切应力 (1 dyn/厘米2) η: 粘度 (0.015 dyn/厘米2) Φ: 流速 (0.67 毫升/分)</li>
	</ol>
</li>
	<li>
白细胞脱粘
	<ol>
<li>对于脱粘, 从细胞悬浮液中切换油管, 通过挤压油管来检测缓冲液, 防止气泡被困住。</li>
		<li>用适当的流速/剪切应力 (见3.3.7 注), 用检测缓冲器分离细胞。</li>
	</ol>
</li>
	<li>
白细胞轮回
	<ol>
<li>对于白细胞轮回, 灌注白细胞 (步骤 3), 直到足够数量的白细胞坚持 (~ 50 细胞/查看领域)。</li>
		<li>用化验缓冲器代替白细胞悬浮液。</li>
		<li>记录 transmigrating 细胞的时间间隔每 10–15 s 为30–60分钟在37摄氏度。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Dinauer, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+immune+deficiencies+with+defects+in+neutrophil+function.">Primary immune deficiencies with defects in neutrophil function.</a> American Society of Hematology. 2016 (1), 43-50 (2016).</li><li>Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+cell+recognition:+three+(or+more)+steps+to+specificity+and+diversity.">Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity.</a> Cell. 67 (6), 1033-1036 (1991).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76 (2), 301-314 (1994).</li><li>Nourshargh, S., Alon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+migration+into+inflamed+tissues.">Leukocyte migration into inflamed tissues.</a> Immunity. 41 (5), 694-707 (2014).</li><li>Legein, B., Temmerman, L., Biessen, E. A. L., Lutgens, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+immune+system+interactions+in+atherosclerosis.">Inflammation and immune system interactions in atherosclerosis.</a> Cellular and Molecular Life Sciences. 70 (20), 3847-3869 (2013).</li><li>Wang, Y., Sakuma, M., 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=Leukocyte+engagement+of+platelet+glycoprotein+Ibalpha+via+the+integrin+Mac-1+is+critical+for+the+biological+response+to+vascular+injury.">Leukocyte engagement of platelet glycoprotein Ibalpha via the integrin Mac-1 is critical for the biological response to vascular injury.</a> Circulation. 112 (19), 2993-3000 (2005).</li><li>Zhao, Z., Vajen, T., 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=Deletion+of+junctional+adhesion+molecule+A+from+platelets+increases+early-stage+neointima+formation+after+wire+injury+in+hyperlipidemic+mice.">Deletion of junctional adhesion molecule A from platelets increases early-stage neointima formation after wire injury in hyperlipidemic mice.</a> Journal of cellular and molecular medicine. , (2017).</li><li>Wang, Y., Gao, H., 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=Leukocyte+integrin+Mac-1+regulates+thrombosis+via+interaction+with+platelet+GPIb&#945;.">Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIb&#945;.</a> Nature communications. 8, 15559 (2017).</li><li>Fujii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS-based+microfluidic+devices+for+biomedical+applications.">PDMS-based microfluidic devices for biomedical applications.</a> Microelectronic Engineering. 61-62, 907-914 (2002).</li><li>Son, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+shear+viscosity+and+shear+rate+from+pressure+drop+and+flow+rate+relationship+in+a+rectangular+channel.">Determination of shear viscosity and shear rate from pressure drop and flow rate relationship in a rectangular channel.</a> Polymer. 48 (2), 632-637 (2007).</li><li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+how+to+generate+and+visualize+them.">Neutrophil extracellular traps: how to generate and visualize them.</a> Journal of visualized experiments : JoVE. (36), e1724 (2010).</li><li>Swamydas, M., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+purification+and+labeling+of+mouse+bone+marrow+neutrophils+for+functional+studies+and+adoptive+transfer+experiments.">Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments.</a> Journal of visualized experiments : JoVE. (77), e50586 (2013).</li><li>Schmitt, M. M. N., Megens, R. T. 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=Endothelial+junctional+adhesion+molecule-a+guides+monocytes+into+flow-dependent+predilection+sites+of+atherosclerosis.">Endothelial junctional adhesion molecule-a guides monocytes into flow-dependent predilection sites of atherosclerosis.</a> Circulation. 129 (1), 66-76 (2014).</li></ol>

作者没有什么可透露的。

此体外检测是一种直接的方法, 用于研究血管炎症过程中白细胞招募的基本机制, 但还有一些关键的问题需要注意。成功执行这项检测的第一项要求是在完整的、汇合的血管或血小板单层上灌注白细胞。这可以通过预先涂覆胶原 i 型的表面来实现。一般来说, 在使用主血管细胞的时候, 用推荐的含胶原酶的分离溶液轻轻地拆下细胞是很重要的 (请参阅材料表), 这种方法不那么严格, 但与胰蛋白酶一样有效。对于血小板单分子膜, 在隔离过程中轻轻地清洗血小板以防止血小板活化和聚集是很重要的。
另一项重要的考虑是保持泵的退出模式的连续性, 用于灌注, 因为不连续导致扰动的流速和剪切条件。因此, 这导致实际剪切应力的不精确性, 随后, 对生理剪切依赖过程的误解。这可以通过定期维护水泵来防止。此外, 对血液灌注过程中的连续性至关重要的是每项实验都使用新鲜注射器。因为橡胶的质地在注射器之内改变以越来越使用和时间在实验之间, 它防止连续撤退。
另一个关键步骤是防止空气在白细胞灌注过程中被困住, 因为空气随后会从表面撕裂细胞并改变剪切条件。这可以防止通过冲洗所有油管之前, 使用超纯水, 然后与化验缓冲区。此外, 当连接油管从化验缓冲器到细胞悬浮, 或从一个条件到另一个, 这是重要的是保持液体接口在联接过程中。这可以通过挤压油管或不改变油管内的液体水平来实现。此外, 灌流液的流体体积应始终足以防止水位降到油管端以下, 从而使空气在系统中被占用。
这种检测的可能限制可能是使用在人工环境中维护的培养细胞,即在一个不准确地模拟体内状态的培养基上培养的塑料表面。虽然理化和生理环境可以精确控制, 培养细胞具有快速的生长速率, 从而增加了遗传变异, 从而使细胞在人群中的异质性增大。此外, 血管或血小板单层仅由单个细胞类型组成。特别是在研究内皮细胞-白细胞相互作用时,即, 在内皮细胞膜上的淋巴细胞的轮回, 基础内皮细胞基底膜和毛细血管的生理相关作用。不能被考虑在内。另外, 通过在本试验中实施 3 d 细胞培养技术, 可以创造多细胞环境, 这可能是有趣的。
综上所述, 以这些观点为基础, 可以有效地将层流法应用于不同的血管炎症模型。特别是, 可以很容易地修改以解决特定的研究问题,即通过改变灌流液的流速、流体粘度或通道高度和宽度来调整剪切应力。特别是, 后者是重要的, 因为变化的流动室尺寸减少流体体积或细胞所需。此外, 不同荧光标记的白细胞, 或特定分子的血管或血小板膜可以用来研究细胞间的相互作用。

在受伤, 炎症, 或感染, 白细胞迅速反应病原体-或损害相关的分子模式 (PAMPs, 抑), 转变成一个活化状态, 并从血液流到炎症和组织损伤部位。白细胞与细胞和分子环境相互作用的能力, 对于它们作为免疫细胞的正确功能是必不可少的, 如白细胞黏附缺陷1所突出的基因紊乱。白细胞黏附一直是在过去几十年的激烈调查的主题, 这导致了白细胞粘附级联在二十世纪九十年代代初2,3的概念。白细胞黏附是由选择素介导的白细胞捕获到内皮细胞引发的, 从而导致了血管表面的滚动。这种滚动使白细胞扫描血管内皮细胞的迁移线索,例如, 趋化因子, 诱导激活的整合。随后, 活化整合介导与内皮配体的结合, 导致坚固白细胞的拘捕。白细胞随后会通过爬行和传播来准备 extravasate, 然后穿透内皮细胞单层和 transmigrating 进入底层组织。标准白细胞级联的基本概念自引入以来基本上保持不变, 有些中间步骤添加了4。然而, 目前还没有阐明参与白细胞招募的许多参与者的确切分子机制和作用, 而白细胞的招募仍然是感染、炎症和 (自动) 免疫领域的一个重要课题。研究。
例如, 在血管炎症性疾病, 如动脉粥样硬化, 增加白细胞招募到血管壁驱动斑块发展。不稳定的动脉粥样硬化斑块可能会破裂, 导致血小板和凝血系统的大规模活化, 随后阻断血管5。这可能导致严重的心血管后果, 如心肌梗死或中风。此外, 在临床上发生内皮剥脱,例如冠状动脉支架术后, 导致大量的白细胞和血小板相互作用于暴露的血管壁内部 (例如, 基质成分和平滑肌细胞) 和与血小板覆盖血管损伤的白细胞。这些相互作用是重要的进一步发展的疾病, 因为单核细胞-血小板相互作用可能驱动新生内膜形成6, 7.此外, 白细胞整合素 Mac-1 (αMβ2) 和血小板 GPIbα介导的血小板-白细胞相互作用最近被确定为小鼠8中血栓形成的新驱动因素。
鉴于人类和动物血液作为研究的白细胞和血小板来源的广泛存在, 以及分离基质分子和白细胞和血管起源的永生化细胞系的广泛存在, 模拟白细胞是可行的。在实验室环境中, 使用特别设计的流灌注室进行相互作用。在过去的几十年中, 许多变种已经被设计出来, 从真空驱动到自粘式灌注室不等。所有变体都有共同之处, 即不动部分 (例如, 培养的血管细胞或基质蛋白) 被组装成一个更大的防漏室, 装有预先定义的通道, 使液体在静止的部分上灌注。此外, 成型技术的进步使得基于硅树脂的定制解决方案的开发成为了9。流化液的粘度和流速以及通道的高度, 主要决定了流量灌注装置的剪切应力特性10。在本文中, 我们提出了一个体外方法来研究在静脉和动脉流机制下白细胞黏附、脱粘和轮回的机制。这里提出的方法的优点是, 它们可以使用普通的摄像机连接荧光显微镜进行, 并且不需要实验者拥有高的技术熟练程度。体外分析可以通过多种方式进行操作 (例如, 添加抑制剂或阻断抗体), 因而适用于不同的血管炎症模型, 并允许对黏附蛋白功能的研究或对特定化合物的评价。

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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Inverted fluorescence microscope e.g. EVOS-FL</td>
			<td style="width:189px;">Life Technologies Europe bv</td>
			<td style="width:163px;"></td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:420px;">Pump e.g. model: 210-CE</td>
			<td style="width:189px;">&#160;world precision instruments</td>
			<td style="width:163px;">78-9210W</td>
			<td></td>
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			<td height="20" style="height:20px;width:420px;">Falcon 35 mm TC-Treated Easy-Grip Style Cell Culture Dish</td>
			<td style="width:189px;">corning</td>
			<td style="width:163px;">353001</td>
			<td></td>
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			<td height="20" style="height:20px;width:420px;">50 mL syringe</td>
			<td style="width:189px;">Becton Dickinson</td>
			<td style="width:163px;">300137</td>
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			<td height="20" style="height:20px;width:420px;">Silicone tubing&#160;</td>
			<td style="width:189px;">VWR</td>
			<td style="width:163px;">228-0700</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Elbow Luer Connector Male</td>
			<td style="width:189px;">Ibidi</td>
			<td style="width:163px;">10802</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Female Luer Lock Coupler</td>
			<td style="width:189px;">Ibidi</td>
			<td style="width:163px;">10823</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Flow chamber</td>
			<td style="width:189px;">University Hospital RWTH Aachen</td>
			<td style="width:163px;"></td>
			<td>Patent DE10328277A1: Baltus T, Dautzenberg R, Weber CPD. Str&#246;mungskammer zur in-vitro-Untersuchung von physiologischen Vorg&#228;ngen an Zelloberfl&#228;chen. 2005.&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Ibidi sticky slide VI 0.4</td>
			<td style="width:189px;">Ibidi</td>
			<td>80608</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Coverslips for sticky slides&#160;</td>
			<td style="width:189px;">Ibidi</td>
			<td>10812</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Collagen Type I, rat tail</td>
			<td>Life Technologies Europe bv</td>
			<td style="width:163px;">A1048301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant Human TNF-&#945;</td>
			<td>Peprotech</td>
			<td>300-01A</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Thrombin Receptor Activator for Peptide 6 (TRAP - 6)</td>
			<td>Anaspec / Eurogentec</td>
			<td>24191</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SYTO 13 green fluorescent nucleic acid stain</td>
			<td>Life Technologies Europe bv</td>
			<td>S7575</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Hanks&#8217; Balanced Salt Solution 10x</td>
			<td>Sigma-Aldrich Chemie</td>
			<td>H4641</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES buffer solution 1M, liquid</td>
			<td>Life Technologies Europe bv</td>
			<td>15630049</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">BUMINATE Albumin, 25%&#160;</td>
			<td>Baxter</td>
			<td>60010</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Water for injection</td>
			<td style="width:189px;">B. Braun</td>
			<td style="width:163px;">3624315</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Calcium chloride dihydrate</td>
			<td style="width:189px;">Merck</td>
			<td style="width:163px;">102382&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Magnesium chloride hexahydrate&#160;</td>
			<td>Sigma-Aldrich Chemie</td>
			<td style="width:163px;">M2670</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Apyrase</td>
			<td>Sigma-Aldrich Chemie</td>
			<td>A6535</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Primary Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:189px;">Promocell GmbH</td>
			<td>C-12203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Endothelial Cell Growth Medium (Ready-to-use)</td>
			<td style="width:189px;">Promocell GmbH</td>
			<td>C-22010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Primary Human Aortic Endothelial Cells (HAoEC)</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">&#160;PCS-100-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Vascular Cell Basal Medium&#160;</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">PCS-100-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Endothelial Cell Growth Kit-BBE</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">PCS-100-040</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Primary Human Aortic Smooth Muscle Cells (HAoSMC)</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">PCS-100-012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Vascular Smooth Muscle Cell Growth Kit</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">PCS-100-042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Human endothelial cell line, EA.hy926</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">CRL-2922</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Mouse endothelial cell line, SV 40 transformed (SVEC4-10)</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">CRL-2181</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Human Monocytic cell line, THP-1</td>
			<td style="width:189px;">DSMZ</td>
			<td style="width:163px;">ACC-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 with Ultra-Glutamine</td>
			<td style="width:189px;">Lonza</td>
			<td>BE12-702F/U1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Mouse monocyte/macrophage RAW264.7</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:163px;">TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), high glucose</td>
			<td style="width:189px;">Gibco</td>
			<td style="width:163px;">11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Accutase</td>
			<td style="width:189px;">Promocell GmbH</td>
			<td>C-41310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Percoll</td>
			<td style="width:189px;">Sigma-Aldrich Chemie</td>
			<td>P1644</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Histopaque 1119</td>
			<td style="width:189px;">Sigma-Aldrich Chemie</td>
			<td>11191</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:420px;">10x PBS</td>
			<td style="width:189px;">Life Technologies Europe bv</td>
			<td style="width:163px;">14200075</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:420px;">BD&#160;Vacutainer Plastic Blood Collection Tubes with Sodium Heparin</td>
			<td style="width:189px;">Becton Dickinson</td>
			<td style="width:163px;">367876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">VACUETTE TUBE 9 ml 9NC Coagulation sodium citrate 3.2%&#160;</td>
			<td style="width:189px;">Greiner Bio</td>
			<td style="width:163px;">455322</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:420px;">HCl/EtOH mixture (1.2 mol/L HCl and 50% ethanol) in water</td>
			<td style="width:189px;">Sigma-Aldrich Chemie</td>
			<td style="width:163px;">Prepare by mixing 0.3L HCl (4 mol/L) with 0.7L of 70% v/v ethanol in a fume hood</td>
			<td></td>
		</tr>
	</tbody>
</table>

laminar flow-based assays to investigate leukocyte recruitment on cultured vascular cells and adherent platelets

近几十年来, 在伤害或组织损伤部位对白细胞的招募进行了调查, 并导致了白细胞黏附性级联的概念。然而, 尚未充分确定参与白细胞招募的确切分子机制。由于白细胞的招募仍然是感染, 炎症, 和 (自动) 免疫研究领域的一个重要课题, 我们提出了一个简单的层流基础的分析研究的基础机制的黏附力, 脱粘, 并在静脉和动脉血流机制下的白细胞轮回。体外分析可用于研究在不同的血管炎症模型中, 白细胞与其细胞伙伴之间相互作用的分子机制。该协议描述了一种基于层流的检测方法, 使用平行流腔和倒置相衬显微镜连接到一个相机, 研究白细胞和内皮细胞或血小板的相互作用, 然后再进行可视化和记录。离线分析。内皮细胞、血小板或白细胞可以用抑制剂或抗体进行预处理, 以确定特定分子在这个过程中的作用。剪切条件,即动脉或静脉剪切应力, 可以很容易地适应的粘度和流速的灌注液和高度的渠道。

为研究内皮-白细胞黏附, 荧光标记 THP-1 细胞在3达因/cm2的 TNFα或非刺激的内皮层中灌注2分钟。通过在2-6 分钟内捕获6个独立的字段, 在2分钟的灌注后确定黏附单核细胞细胞总数。在至少6张用倒置相对比/荧光显微镜 (例如, EVOS) 拍摄的图片中, 用10X 放大器连接到数字 CCD 或 CMOS 相机, 并将平均值表示为粘附细胞/mm2。TNFα内皮细胞刺激后, THP-1 的骤停与静态内皮明显增高。在图 1中, 有代表性的显微图和对非或 TNFα刺激内皮细胞的牢固黏附 THP-1 的量化。在1达因/cm2上, 用荧光标记的中性粒细胞在 TRAP-6 或非刺激的血小板单层上灌注2分钟, 评估血小板-白细胞黏附力。粘附中性粒细胞的数量如上文所述。TRAP-6 刺激显著增加了中性粒细胞相对于静态血小板的黏附力。在图 1B中, 有代表性的视频和对非或 TRAP-6-stimulated 血小板单层牢固黏附的中性粒细胞的量化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57009/57009fig1.jpg"> 图 1: 流条件下内皮和血小板-白细胞黏附.定量和代表性的显微照片的人单核细胞细胞 (THP-1) 对内皮细胞 (HUVEC) 播种在胶原涂层培养皿在流动条件下, 有或不 TNFα活化 (10 ng/毫升)(A)。在流动条件下, 无或与 TRAP-6 激活 (50 µM) (B) 的胶原涂层玻片固定化的人中性粒细胞与人血小板黏附的定量和代表性显微照片。P 值由配对 t 检验计算 (n = 3; 平均值)。缩放条 = 100 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57009/57009fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
此外, 该方法可用于研究黏附分子的作用, 如血小板交界黏附分子 a (果酱 a)。我们的前研究7评估了 RAW264.7 鼠单核细胞/巨噬细胞与果酱 a (trJAM-++) 和果酱 a 缺失 (trJAM-/) 小鼠的粘附。如上所示, 血小板堵塞--一种缺陷增加了小鼠单核细胞与野生型小鼠的黏附力 (图 2A B)。或者, 主老鼠中性粒细胞可以与老鼠隔离, 如12所述。
为了研究潜在的机制, 如粘附分子参与血小板-白细胞相互作用, 特定的表面受体可以阻止。在上述研究中, 阻断血小板 GPIbα和白细胞αMβ2显著降低单核细胞的黏附力, 确定 GPIbααMβ2轴在增加的核细胞招募到果酱中的作用-一个/-血小板 (图 2A-B)。此外, 细胞脱离实验可以用来研究血小板-白细胞相互作用的剪应力依赖性。为了测量单核细胞的流依赖性分离, 剪切应力从0增加到20达因/厘米2。在这项研究中, RAW264.7 细胞在果酱上的黏附力--一个/血小板比果酱更抗剪应力--一个++血小板 (图 2C)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57009/57009fig2.jpg"> 图 2: 果酱 A 缺乏促进耐流单核细胞细胞黏附力。小鼠单核/巨噬 RAW264.7 细胞与血小板的黏附力-一个++和果酱-一个/小鼠固定在胶原涂层的玻璃幻灯片在流动条件下, 有或不凝血酶活化 (IIa, 0.5 毫微米) 存在指示抑制剂 (A)。用同种控制或抗 GPIbα抗体治疗后, 小鼠单核/巨噬细胞 RAW264.7 单元的代表性显微照片粘附在果酱上-一个++和果酱-一个/血小板 (B)。在凝血酶活化后固定化血小板 (IIa, 0.5 nM) 下, 细胞黏附力表达为最初黏附细胞在增量性增加的剪切应力 (达因/厘米2) 上的百分比。P 值是通过方差分析计算的 Tukey 的后测试 (n=3-5; 平均电子扫描电镜)。刻度条 = 100 µm。此数字已从赵et 等中修改。7<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57009/57009fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
在白细胞灌注和粘附后, 单个磁场的持续记录时间 (30–60 min) 允许研究白细胞爬行和传播行为以及在内皮层上的最终轮回。在这项研究中, 我们分析了第一人单核细胞的轮回 (与单核细胞隔离套件隔离, 根据制造商的说明, 如13所述) 横跨内皮层。如图 3所示, 在对内皮细胞进行严格的逮捕后, 单核细胞通过爬行和传播来准备 extravasate, 直到到达首选的轮回地点。随后, 单核细胞通过准或 transcellular 迁移, 穿透内皮细胞屏障。Transmigrating 细胞被定义为分散在内皮细胞上的细胞, 通过扩展它们的伪足和通过内皮屏障, 从而降低其亮度。
<img alt="Movie 1" src="/files/ftp_upload/57009/57009mov1.jpg"> 图 3:体外将主单核细胞在内皮层间轮回.单核细胞爬行和传播, 并在整个内皮细胞的最终轮回记录每十五年代30分钟.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57009/Transmigration_short.mov" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>

Watch this Scientific Journal Video about Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets at JoVE.com

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

在血管壁损伤或炎症期间, 白细胞与血管细胞和血小板相互作用。在这里, 我们描述了一种简单的基于层流的检测方法来表征白细胞与其细胞伙伴之间相互作用的分子机制。

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

laminar-flow-based-assays-to-investigate-leukocyte-recruitment-on

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.5K 次观看.  Maastricht University. 在血管壁损伤或炎症期间, 白细胞与血管细胞和血小板相互作用。在这里, 我们描述了一种简单的基于层流的检测方法来表征白细胞与其细胞伙伴之间相互作用的分子机制。

视频: Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

成像白细胞粘附到血管内皮细胞在高腔内压力

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

科研

免疫与感染

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

昆虫<em&gt;大蜡螟</em作为一个强大的的感染模型探讨细菌致病

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

猪或牛光感受器外段的细胞吞噬分析对视网膜色素上皮细胞大规模纯化

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

应用系统进化分析研究真核基因的起源

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...