这项工作是由国立法新社德拉RECHERCHE（Chercheuses的Jeunes / Chercheurs的Jeunes到EFN），基金会VOIR等Entendre和贝当古基金会舒尔勒（青年研究补助金，以EFN），中心法国国家科学研究（CNRS，为EFN永久位置）支持和健康（R01-EY13295到SCF）全国学院的国立眼科研究所。此外，研究所德拉视觉是由国家研究所德拉桑特等德拉RECHERCHE MEDICALE，UNIVERSITE皮埃尔与玛丽居里 - 巴黎6，中心法国国家科学研究和百货巴黎资助。

这个POS隔离实验是耗时且可能需要长达12小时来完成，如果POS存储之前被标记。该协议是改编自1987年31 1997年10由RS Molday和同事发表和修改SC Finnemann和同事的论文。 动物根据研究协会视觉与眼科（ARVO）声明的眼科和视觉研究使用动物的处理。协议进行了审查并批准了查尔斯·达尔文的道德委员会从大学的皮埃尔和玛丽·居里 - 巴黎06和Fordham大学机构动物护理和使用委员会。 1.一般设置<ol><li>屠宰尽可能获得后新鲜一旦80鲜猪或牛的眼睛，让他们冷冻在黑暗中。提供指令给供应商，以便该公司进行的方式相同。该协议是优化80眼。到进行更多的目光并获得更大的股票（ 例如，从160眼），双液量并进行2轮超速离心的;游泳池样品后来在收集步骤（见步骤2.2.2）。 </li><li>设置在板凳上的红色安全指示灯，吸水垫，湿巾（ 材料表 ）。准备15厘米菜肴和生物危害袋垃圾收集（未用眼睛部位，玻璃体...）。穿紧身labcoat，手套，护袖和护目镜。 </li><li>注意事项： <ol><li>预冷所有的解决方案，然后在所有的步骤让他们感冒。保持眼睛和梯度冷冻冰尽可能。 </li><li> 保持在黑暗中尽可能直到超速离心后收集步骤，以避免光漂白视色素和感光色素的活性形式的橙粉色的丧失的眼睛和衍生组织 。 </li></ol></li></ol> 2.协议操作<ol><li> 准备解决方案和渐变： <ol><li>解冻牛磺酸股票完全采用37℃水浴。 </li><li>准备从原液所有解决方案（ 表1，储备溶液），并充分混合，用磁力搅拌器对每个溶液中的其它成分的蔗糖。 <ol><li>制备15ml的所述均质溶液至20％蔗糖的最终浓度，20毫摩尔Tris pH7.2的醋酸盐，2mM的MgCl 2的，10mM葡萄糖和5mM牛磺酸。 </li><li>制备25％的蔗糖溶液，以25％的蔗糖（使用70％的蔗糖的库存），20毫摩尔Tris乙酸盐pH为7.2，10mM葡萄糖和5mM牛磺酸的最终浓度。 </li><li>制备的60％的蔗糖溶液，以60％的蔗糖（使用蔗糖粉末）的终浓度，20毫摩尔Tris pH7.2的醋酸盐，10mM葡萄糖和5mM牛磺酸。 </li><li>制备洗涤溶液1至20毫摩尔Tris乙酸盐pH为7.2和5mM牛磺酸的最终浓度。 </li><li>准备洗2解决方案至10％的蔗糖，20毫摩尔Tris乙酸盐pH为7.2和5mM牛磺酸的最终浓度。 </li><li>制备洗3溶液至10％的蔗糖，20mM磷酸钠pH为7.2和5mM牛磺酸的最终浓度。 </li></ol></li><li>平稳梯度制造者磁力搅拌器上并插入在最靠近其中60％的溶液将被插入所述出口腔室中的搅拌棒。使用的截止P200吸管尖放置在管道的出口处，以达到适当的铸造速度。 </li><li>微妙的稀释和搅拌与一个25％使用12毫升每个解决方案（ 即共6管80眼）预冷超速离心管的60％的蔗糖溶液浇铸线性渐变。搅逐渐-稀释60％的蔗糖溶液好。平稳梯度流的速度，浇注溶液顺畅且不能太快。 注意：为了得到一个连续的，线性梯度，保持吸管尖开口就在整个梯度formatio溶液的表面ñ。 </li><li>让梯度坐在冰上约1小时的稳定。保持冷冻，直到加载的梯度。不要摇晃试管，防止梯度的破坏。 </li></ol></li><li> 组织收集： <ol><li>收集在昏暗的红灯组织。取的眼睛成一方面与戳前端与刀片边缘，同时保持眼距（以避免飞溅; 图1）。使用剃刀刀片切割的前眼球成两半（角膜加上至少5mm到巩膜）。取下透镜和里面翻转眼罩出来，使得它可以保持在一个手指从而暴露在视网膜的小费。 </li><li>使用剃刀刀片以一个角度，轻轻刮去视网膜，拆卸容易，并显示为粉红色层，脱绒毡层表面和切断视神经头（ 图1）。收集所有的视网膜中2×50毫升各含15ml中在冰上均化溶液管中。 </li><li> 视网膜同源genization： <ol><li>为了扰乱不同的细胞层，折断的POS落公关细胞，片段的POS的其余部分，均质视网膜悬浮摇动该悬浮液剧烈用手2分钟。 </li><li>滤波器3倍通过双层纱布清洁，以除去大的组织片段，并收集流过清洁50ml试管（ 图1）。视网膜悬挂是很厚，最大化收益，轻轻按下每个纱布过滤后释放剩余的零散组织。 </li></ol></li></ol></li><li> 光感受器外段片段（POS）隔离： <ol><li>轻轻地将粗视网膜准备，约6 - 7毫升每管中，在6×30毫升超速离心管各包含24个毫升新鲜冷冻连续25 - 60％的蔗糖梯度（ 图1）。平衡相对管以适合所使用的转子。超速，在106000 XG为50分钟，在4℃。 </li&gt; <li>吸（ 图2A）去除大部分上面的橘粉色的带坡度的上三分之一的解决方案。用一个截止P1000尖到烧杯中收集橙粉色带。使用漂白剂（生物垃圾）中和后，放弃休息。 </li><li>稀释〜4 - 5卷冰冷的洗涤1解决方案。分成许多30毫升管需要。离心机在3000×g离心在4℃下10分钟。小心弃去上清液。 </li><li>在10毫升重悬粒料洗净2溶液并旋在3000×g离心10分钟，具体如上所述。在15毫升重悬粒料洗净3，并再次旋转以3,000×g离心10分钟。组合来自几个粒料悬浮，以减少在洗涤2离心前需要管的数目和清洗3的解决方案。 </li></ol></li><li> POS等分无标签： <ol><li>悬浮POS 10 - 20毫升DMEM。预稀释10微升在490微升DMEM（1:50）和计数细长以及在一个旋转着的POS细胞计数室（ 图2B）。计算产量和浓度在每毫升的POS颗粒。 注：产量变化，取决于主要的年龄和应变的猪/奶牛和眼睛的大小， 例如，良好的制剂的范围内，从5 - 8×10 9的POS 80的猪眼。 </li><li>决定最终的DMEM体积上（一般为10 - 20ml）中，并添加蔗糖以产生2.5％的蔗糖的最终浓度。 </li><li>制备所需大小的等分试样， 例如，每管5×10 7的POS为2ml的悬浮体积为96孔试验，在40微升/孔。自旋1等分试样5分钟，在2300×g离心在RT和评价粒料尺寸。准备各种尺寸的试样为不同的应用量为POS不应被冻结，解冻两次。 </li><li>冻结POS等分在-80°C，直到进一步使用。 注：等份稳定许多个月。在我们手中，未标记的或标记的POS可以存储至少一年。 </li></ol></li><li> POS标记和分装 ： 注意：我们通常使用的FITC标记的POS为吞噬作用测定法的定量，但其它的染料也可以使用，以及根据需要。 <ol><li>重悬1 10毫克的FITC小瓶在2.5毫克/毫升的浓度在0.1M碳酸钠缓冲液pH 9.5通过混合2.6毫升的0.1M的NaHCO 3 pH8.4的与1.4毫升的0.1M的Na 2 CO 3的pH 11.5（ 表2，储备溶液）。旋转1小时在室温下，同时从光及自旋保护5分钟，在最大速度下，以沉淀的非重悬于FITC的固体。 </li><li>重悬在POS沉淀在10ml洗涤3溶液（或DMEM）中，并加入2毫升FITC溶液80的眼睛。存储剩余FITC冷冻在-80℃。旋转为至少1.5小时，在室温。用铝箔避光。 </li><li>直到几乎没有游离的FITC被认为是在上清液F之类在第2.3.4节中所述洗净用洗净3溶液两次标记的POS，然后一次或两次的DMEMraction。悬浮在POS DMEM并继续为未标记的POS计数和分装（ 第2.4节 ）。 </li></ol></li><li> 在实验中使用POS： <ol><li>解冻的POS在RT，并保持在黑暗中尽可能如果它们是荧光标记的。旋转5分钟，在2300 XG在室温。吸出上清液。 </li><li>立即悬浮在测定溶液适当体积决定了实验。的情况下的POS挑战不同的时间，存储在时间点之间的暗的POS悬浮于室温进行数小时。 </li></ol></li></ol>

The authors declare that they have no competing financial interest.

三个步骤或条件是一个优化的纯化至关重要：梯度铸造细腻梯度管操纵质量，保持组织冷冻并在黑暗中直到收集步骤，抖视网膜匀浆的强度，从PR的其余部分获得适当的POS隔离细胞。如果有些问题出现在正确看到橙色的乐队，他们是由于三个原因大于1（另见结果部分的第二段），最有可能的。某些条件做一些修改是可能的，就像使用27 - 50％的蔗糖浓度梯度32或Optiprep步梯度<sup...

在视网膜，视力是由光敏分子，称为视蛋白，被转化成可在神经元之间向上传送到视觉区域在大脑中的信号之前的异构触发。这些分子被嵌入在叠层膜的磁盘类似，构成感光细胞外节的部分（PRS）的煎饼。受到恒定曝光，因此，相当大的水平的氧化应激，收受不断更新它们的外节段，以限制潜在的氧化损伤。光感受器外节是与相邻的视网膜色素上皮（RPE）细胞的顶端微绒毛紧密接触。视网膜色素上皮细胞构成的血-视网膜屏障的外部部分，并确保许多任务，这对于光感受器的健康和功能1是至关重要的，例如经由黑色素颜料，再异构化光反应视蛋白成分的视网膜，提供营养物和克淬火光线rowth因素，参与PR​​代谢产物处置。 此外，RPE细胞消除花费POS和回收它们的组件，一个由昼夜节律在哺乳动物视网膜2,3调节每日占领。棚的POS的间隙为PR生存绝对必要的。当它被完全废除，POS碎片累积和永久退化造成快速视力下降4,5。如果有节奏的配置文件丢失，通过不断的活动取代，PR和RPE缺陷积累与6岁。因此，这是非常重要的，以便了解表型与它的功能障碍以表征RPE吞噬作用的分子调控体外 。有趣的是，在视网膜色素上皮细胞的分子机制非常类似于使用由巨噬细胞清除凋亡细胞的1和两者都依赖于识别暴露磷脂酰丝氨酸上吞噬碎片7-9。仍然，RPE细胞和巨噬细胞调节pH值agocytosis不同，如巨噬细胞相遇时选择立即消除凋亡细胞的同时RPE细胞有节奏，每天吞噬POS只有一次，尽管他们与外节永久接触。这表明，还没有完全理解特定调节机制。 许多牵连的视网膜色素上皮细胞吞噬机械的分子已被确定或确认由于使用分离的POS和细胞培养物的吞噬作用测定法。与其配体MFG-E8位于RPE顶端细胞表面上的alphavbeta5整联蛋白受体，在协调，特异性结合的POS 10-12，其然后经由MERTK酪氨酸激酶受体13-15内在化。的CD36的清道夫受体已经显示参与POS摄取并影响其速度16,17，和可作为氧化磷脂在POS表面18的传感器。内在需要F-肌动蛋白细胞骨架屁股招聘ociated蛋白例如膜联 ​​蛋白2 19，肌球蛋白II 20和肌球蛋白VIIA 21,22。 体外天然或氧化的POS也利用来理解RPE细胞的体内连接于消化的不良氧化的POS 23-28中累积的衰老表现型。从干细胞的RPE细胞的产生已经开始对被用来证明细胞的功能它们被移植到动物或患者29,30,27之前孤立的POS一个新的应用程序。 首先由Molday和他的同事在1987年31所述，协议牛POS隔离结合连续蔗糖梯度与观察漂白视网膜感光色素的特点橙色外观的视网膜匀浆的超速离心步骤（携带11- 顺式视黄醛）。在过去的10年里，由于为了减少疯牛病的风险采取的预防措施，利用猪的眼睛已成为increasingly突出。这里所描述的协议说明如何获得从猪或牛的眼睛可以被等分并储存延长的时间段的大量的POS的。这样就无需从啮齿动物的眼睛，这需要使用大量POS每个准备和检测32,33,21,22动物准备POS。另外，使用荧光分子贮存前约POS标注细节给出，量化和可视化的POS中用于相对于吞噬测定32,10后的POS标签一些应用的简化和可比的方式。因此，这些大股允许再现性和易用性在许多不同类型的实验。

<table border="0" cellpadding="0" cellspacing="0" width="811">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:311px;">Name of Material/ Equipment</td>
			<td style="width:108px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Specific Material/Equipment</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">2-chamber gradient maker</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td>gradient maker with 30-ml chambers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">3-mm diameter silicone tubing</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td>tubing for gradient casting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">small size magnetic stir bar</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td>stir bar fitting the gradient maker chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">red safelight lamp</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td>inactinic lamp for dissection in the dark</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Clear 25x89 cm tubes</td>
			<td>Beckman</td>
			<td>344058</td>
			<td>ultracentrifugation tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PP Oak Ridge tubes</td>
			<td>Nalgene</td>
			<td>3119-0050</td>
			<td>30-mL centrifugation tubes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optima LE-80K&#160;</td>
			<td>Beckman Coulter&#160;</td>
			<td>365668</td>
			<td>ultracentrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW 32Ti swing rotor</td>
			<td>Beckman Coulter&#160;</td>
			<td>369694</td>
			<td>swing rotor for ultracentrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avanti J-26 XP</td>
			<td>Beckman Coulter&#160;</td>
			<td>393124</td>
			<td>centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JA-25.50 rotor</td>
			<td>Beckman Coulter&#160;</td>
			<td>363058</td>
			<td>rotor for Avanti J-26 XP centrifuge</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">FITC Isomer I</td>
			<td>Life Technologies&#160;</td>
			<td>F-1906</td>
			<td>fluorescent dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Other Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">counting chamber (such as Neubauer or Malassez)</td>
			<td style="width:103px;"></td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dark ice buckets with lids</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">scales</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic stirrer and upholding pole</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">refrigated microcentrifuge</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37 &#176;C water bath</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">-80 &#176;C freezer</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">labcoat</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td>Health and safety</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gloves</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sleeve protectors</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">googles</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">absorbent pads</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">biohazard trash bags and bins</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Weck-Prep blades (60-mm/2.25-in wide razor blades)</td>
			<td style="width:103px;"></td>
			<td>Dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm plastic dish</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile gauze sheets</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15- and 50-mL tubes</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td>Common consumables</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microtubes</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aluminum foil</td>
			<td style="width:108px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells

可以在体外进行的视网膜色素上皮（RPE）细胞，光感受器外节（POS）的花费老化远端片段的吞噬的重要功能之一的分析。光感受器外节与栈含有光转机械膜质盘被连续更新的在视网膜上。花了POS是由视网膜色素上皮细胞每天淘汰。啮齿动物，猪/牛和人RPE细胞识别以类似的方式，从不同物种的POS。为了便于进行大系列很少变性的实验中，一个大的股票的POS可以从猪眼来分离和冷冻储存在等分试样。该协议需要时蒙在鼓里中显示橙色的感光色素的特性优势。在昏暗的红灯，视网膜被收集在从切成两半打开眼罩的缓冲区。视网膜细胞悬浮液均化，过滤，并上样到一个连续的蔗糖梯度。岑后trifugation，POS位于一个离散的频带中具有特性橙色渐变的上部。 POS然后收集，纺丝，在洗涤缓冲液再悬浮依次计数并等分。可用于吞噬作用测定法和蛋白激活，定位或相互作用的POS激发后的不同时间分析得到这样的POS。此外，POS可以标记荧光， 例如 ，FITC，等分的POS随后的荧光定量结合或吞噬之前。其他可能的应用包括使用改性的POS或POS挑战结合胁迫条件，研究氧化应激或RPE细胞老化的作用。

线性蔗糖梯度和超速离心的组合允许视网膜悬浮液密度的不同组分的分离。重较大的视网膜碎片和RPE细胞沉到或接近渐变（ 图2A）的底部。来自视网膜打火机POS和较轻的单个细胞或细胞碎片迁移作为单独的频带通过离心期间结束到达梯度的上半部分。通过保持眼睛和样品在黑暗中直到离心步骤后，含有的POS频带可立即通过其明亮的橙色颜色识别。该频带的宽度和强度取决于梯度质量?...

Watch this Scientific Journal Video about Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells at JoVE.com

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

large-scale-purification-porcine-or-bovine-photoreceptor-outer

Research

JoVE Journal

Immunology and Infection

11.9K 次观看.  INSERM, U968. 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 °C.

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

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

淋巴细胞微粒和检测其促凋亡作用的气道上皮细胞产生

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

细胞凋亡细胞的吞噬作用<em&gt;果蝇</em&gt;胚胎

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

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

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

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

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

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

从人类唾液腺中分离唾液表皮细胞,作为脂质层或单层体进行体外生长

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...