作者要感谢Nandini Nallappan和Sharon Yeo帮助技术步骤。研究由南洋理工大学李孔ian医学院KGC的初创资助和新加坡卫生部国家医学研究理事会（NMRC）至LT（NMRC / CSA / 045/2012），并由国家卫生研究中心授予KGC和LT（NHIC-12D-1409007）国家卫生创新中心的资助。

本研究中使用的所有眼部样品均由新加坡国家眼科中心的眼科诊所收集，由SingHealth集中制审查委员会和新加坡南洋理工大学机构审查委员会批准。 注意:受试者进行临床测试以评估炎症的程度和IC执行前泪液功能障碍的严重程度。临床试验包括用诊断仪器评估的非侵入性撕裂破裂时间（NI-TBUT） 14和结膜发红（充血） 15,16 ，Schirmer测试17 ，眼睛干燥的标准患者评估（SPEED）问卷18和角膜染色19 。 IC收集眼睛样本<ol><li>确定临床状态后，麻醉通过施用1-2滴的局部麻醉，盐酸普拉卡因，上肢结膜和下穹窿，参与者的眼睛。等待4 - 5分钟，麻醉剂的刺痛感消失。 </li><li>从两个印象细胞学装置收集来自鼻和颞结节结膜的样品。 注意:一个设备用于收集两个印象样本。在这里，使用两个装置来收集四个印模膜。 <ol><li>将印象细胞学装置切向地置于结膜上。轻轻按下按钮，按住2 - 3秒。 注意:设备附带膜。按下按钮后自动释放压力。释放压力只需几秒钟就可以移除设备。 </li></ol></li><li>将膜放入含有1mL培养基的微量离心管（Roswell Park Memorial Institute培养基（RPMI）+ 10％胎牛血清（FBS）+ 1％青霉素 - 链霉素（Pen Strep））。 </li><li>通过用10μL移液管尖端连续刮擦约1分钟，将细胞从装置的膜上分离出来。 注意:当膜的表面变得不均匀时，刮擦完成。来自每个膜的细胞数量是可变的（100-500个细胞/膜）。在收集的2 - 3 h内处理样品。 </li></ol> 2.流式细胞仪免疫分型细胞<ol><li>在室温下将细胞以400xg离心5分钟以除去培养基。用50μL流式细胞仪染色缓冲液（磷酸盐缓冲盐水（PBS）+ 0.05％牛血清白蛋白（BSA））重悬细胞沉淀。 </li><li>具有一组抗体的染色细胞（ 例如 ，CD3亮紫（BV）510（UCHT1），CD4别藻蓝蛋白-H7（APC-H7）（SK3），CCR7藻红蛋白（PE）-A，CD45RO PE-花青素7（PE- Cy7）-A，活/死细胞标记7-ADD）在室温下20-30分钟黑暗。根据制造商的方案直接从库存中使用抗体。 注意:对于抗体浓度，使用2.5μLCD3-BV510，1.25μLCD4-APC-H7，CD45RO-PE-Cy7,7-AAD和10μLCCR7-PE。使用外周血单核细胞（PBMCs）滴定不同浓度的抗体。对于滴定，采取制造商的方案中提及的抗体浓度，并使用推荐浓度的一半和四分之一。使用最低浓度的抗体避免溢出到其他荧光染料通道。用所提及的抗体浓度观察到清除信号。补偿是通过首先根据Williams 等人提到的方案测试PBMC进行的。 20对于测试，将PBMC与本文使用的相同抗体组合孵育，并通过比较单次和多次抗体染色来补偿。 </li><li>孵化周期后od，向管中加入1mL染色缓冲液，充分混合，并在室温下以450xg离心5分钟。将细胞悬浮于200μL染色缓冲液中。 </li><li>在流式细胞仪上运行样品。 <ol><li>在获取数据之前，请使用细胞仪设置和跟踪（CS＆T）珠校准流式细胞仪通道电压，以按照制造商的说明在不同日期对数据采集进行归一化。 <ol><li>打开流式细胞仪数据采集软件，点击"设置＆QC"按钮，选择正确的CS＆T珠批号，加载珠，然后点击"开始"按钮。 </li></ol></li><li>在将样品加载到机器前，轻轻敲打管，重悬细胞。打开数据采集软件，点击"预览"。当阈值稳定时，单击"获取"。监控样品级别，当样品用尽时点击"停止"。 注意:每个样品获取10,000个事件。 </l我&gt; <li>获取10,000个事件后，在工作表中生成SSC-A和FSC-A点图。 <ol><li>单击"多边形门"按钮，在FSC-A值&gt; 5 x 10 4的所有事件上绘制一个门（这是P1），以排除碎片。点击"创建点图"按钮生成一个新的点图，右键点击斑点，选择"属性"打开"绘图编辑器"窗口，然后选择"P1"。点击P1点图的x轴上的FSC-A，将其更改为CD3-BV510。画一个"多边形门"来选择CD3 +群体并命名为"P2"。 </li><li>点击"创建点图"按钮生成一个新的点图，右键点击斑点斑点，选择"属性"打开"绘图编辑器"窗口。点击P2点图的x轴上的FSC-A，将其更改为7-AAD。为7-AAD -人口绘制"多边形门"，并选择"P3"。 P3这里是CD3 +活细胞</li><li>点击"创建点图"按钮生成一个新的点图，右键点击斑点斑点，选择"属性"打开"绘图编辑器"窗口。点击P3点图的y轴上的x轴上的CD3-BV510和CD4-APCH7。在象限的上下两边绘制矩形门，并分别选择"P4"和"P5"。 P4是活CD3 + CD4 + ，P5是活CD3 + CD4 - T细胞。 </li><li>点击"创建点图"按钮生成一个新的点图，右键点击斑点斑点，选择"属性"打开"绘图编辑器"窗口。单击x轴上的CD45RO PE-Cy7和P4和P5图的y轴上的CCR7-PE。在这个点图上画"四门"。 注意:这里的左上侧，右上侧，右下侧和左下方的象限是天真的，中央记忆（T CM ），effector记忆（T EM ），终末分化的效应记忆（T EMRA ）T细胞。 </li></ol></li></ol></li></ol>

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作者宣称没有竞争的经济利益。

与常规技术（如刮擦，擦拭，移液或吸收性滤纸1,2）相比 ，这是一种容易，快速和较少入侵的技术，可用于门诊诊所相对较快的免疫分析。这种技术的一个变体已经在研究设置22中被使用。拟议方法的未来应用是在眼部疾病的临床试验中进行患者分层，特别是需要免疫表型分析的患者。 这种技术的主要挑战是在印模收集和刮除之后检索到的相对较少的免疫细胞。从每个个体的四次印迹中回收的CD3 + T细胞的总数从约500-1000个细胞变化。在流式细胞术分析前将眼睛样品洗涤最少次，以避免细胞进一步损失秒。保留在协议中的关键步骤和挑战是高效收集眼睛样品并适当刮擦膜以达到更高的细胞数量。然而，这种限制不太可能偏向任何特定的免疫表型。在这里进行的用于最大化细胞产量的故障排除是减少抗体孵育之后和之后的洗涤步骤的数量。 使用IC还有其他限制。在严重角化或纤维化的眼表面如Steven Johnson综合征患者中，细胞产量可能甚至低于本研究中的。如果样品被储存，而不是在同一天进行分析，免疫细胞的比例可能会改变。难以预测某些细胞类型是否比其他细胞类型更耐贮存。以前的研究报道了结膜上皮细胞23中HLA-DR表达水平升高，因此它将是inter评估HLA-DR与特异性免疫细胞水平之间的相关性。免疫细胞也可能与趋化因子的表达水平相关。这些问题应在今后的研究中得到解决。

印度细胞学（IC）于1977年由Thatcher等人 1首先进行。他们使用塑料印版光盘从患者收集结膜细胞，而不是当时可用的其他技术，如刮擦，擦拭或移液1 。目前的IC技术使用吸收性滤纸2印刷延髓和睑结膜并收集最表层的结膜细胞。这些细胞已达到分化的最后阶段，不断流下眼泪3 。在IC标本中发现三种主要的细胞群:上皮细胞4 ，杯状细胞3,5和粘膜相关淋巴组织viz上皮相关的效应T细胞或树突状细胞6 。 IC中的眼表细胞可以通过显微镜，免疫印迹和逆转录酶聚合酶链反应（RT-PCR）进行分析。流式细胞术最近被用来分析刮擦IC膜8所收集的免疫细胞。有趣的是，IC6,9已被用于评估许多眼表疾病，包括角结膜炎西卡，维生素A缺乏症，瘢痕性类天疱疮，特应性疾病，上限角膜结膜炎，春季角膜结膜炎和上皮鳞状上皮化生。 IC还被用于评估在纵向研究10,11,12中佩戴隐形眼镜，检测眼表面微生物以及治疗干预措施的疗效和耐受性的影响。 医疗设备（EyePrim）由一种聚合物支持已经使用流式细胞仪（OSIC-flow）先前验证了用于眼部印象细胞学技术的醚砜（PES）0.2μm膜，并且开辟了使用纵向采样来监测疾病进展和对治疗反应的机会（ 例如详细上皮细胞白细胞的分析被定义为粘膜性天疱疮进行性结膜纤维化的推定性疾病标志物。早期研究人员使用需要人工印象的高压灭菌PES过滤器。因此，产量是可变的和用户依赖的。该医疗设备的优点是易于使用，标准化压力（Pa或N / m 2 ），并且具有重复性，可重复性和一致的细胞恢复。这种技术在门诊诊所中非常有用，因为它是非手术的，易于执行的和快速的。这是根据指令93/42 / CEE，CE 0499（SNCH）的I类（无菌）医疗器械。它只需要在手术过程中的局部麻醉，确保维持眼表的完整性。在IC后，可以立即对细胞进行流式细胞术处理。此外，可以对非眼科技术人员和护士进行训练以对眼表进行采样。 尽管IC与其他技术有所改进，但仍然存在若干挑战。例如，取决于IC的位置，取样面积和球形结膜的区域差异可能会有变化。变化的另一个来源是由于IC中应用了不同的压力。其他方法问题涉及细胞处理的标准化:这些涉及持续时间和固定方法以及可能存储的条件，这可能影响采样材料的稳定性。 该技术的总体目标是开发一种隔离眼睛印象样本的方法t易于使用，非侵入性，可应用于临床样品的免疫学表征。

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			<td height="21" style="height:21px;width:331px;">Reagents</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">anti-human CD3 BV510</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">563109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">anti-human CD4 APCH7</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">641398</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">anti-human CD45RO PECy7</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">337168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">7-AAD solution</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">555816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">anti-human CCR7 PE</td>
			<td style="width:300px;">BD Biosciences</td>
			<td align="right">552176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Pippetes</td>
			<td style="width:300px;">Eppendorf</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Local Anaesthesia</td>
			<td style="width:300px;">Alcaine</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fluorescein sodium solution</td>
			<td style="width:300px;">Bausch &#38; Lomb U.K Limited</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:331px;">Name</td>
			<td style="width:300px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Equipments</td>
			<td style="width:300px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Keratograph 5M</td>
			<td>Oculus</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Slit lamp BioMicroscope</td>
			<td>Haag Streit</td>
			<td>BM900</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">EyePrim</td>
			<td>Opia Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FACS Verse</td>
			<td style="width:300px;">BD BioSciences</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:331px;">Name</td>
			<td style="width:300px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Softwares</td>
			<td style="width:300px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">GraphPad 6.0</td>
			<td style="width:300px;">Prism</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FACSVerse analysis software</td>
			<td style="width:300px;">BD</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

a non-invasive way to isolate and phenotype cells from the conjunctiva

传统上，用诸如刮铲技术和刷技术的技术来研究眼表细胞学。这些技术的问题在于，它们可以在眼睛表面上诱发创伤性损伤，这可能发展成瘢痕，眼睑畸形，角膜缘干细胞缺乏，并且在某些情况下，对受试者造成极大的不适。为了避免这些临床问题，开发了印象细胞学（IC）以诊断干眼病和后期肿瘤，特应性疾病，春季角膜结膜炎和角结膜炎。通常，临床医生手动将滤纸切割成所需的形状并将其应用于眼表面。在这里，我们描述如何使用市售的医疗设备来执行IC。这里解释了这种技术，然后通过流式细胞术进行免疫表型分析。这种技术需要较少的手动处理，并减少对眼表的伤害。

通过该临床装置的IC允许我们分离眼表面免疫细胞，同时保持眼表完好无损。 图1描述了IC的执行情况。标记样品收集的眼睛的不同部位。 图2显示了从10个健康对照收集的流式细胞术的代表性结果。对于门控，使用SSC和7-AAD来区分活细胞和死细胞。 7-AAD-细胞被鉴定为活细胞。这些活细胞的进一步特征是CD3 +和CD4 +标记。此外，CD4 +和CD4-群体每个进一步被表征为具有CCR7和CD45RO标记的天然，T EM ，T CM和T EMRA 。在CD3 + T细胞中，效应记忆T细胞在人眼表面占主导地位。在早期e实验中，也证明CD8 +记忆细胞是健康个体中结膜上皮T细胞的主要群体20 。 图3显示，在10个健康对照研究中，CD4 +和CD8 +效应记忆T细胞（T EM和T EMRA ）是人眼表面的主要亚组。图中提到的每个人群都以其标志物表型和名称（Naïve，T CM ，T EM ，T EMRA ）通知。红色的数字表示人口的百分比。该图中的数据表示为平均值±SEM <img alt="图1" class="xfigimg" src="/files/ftp_upload/55591/55591fig1.jpg" /> 图1:通过印象细胞学装置从眼表收集IC样品。从颞叶收集样品ar区域如图所示。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55591/55591fig2.jpg" /> 图2:使用流式细胞仪的斑点图。选择活7-AAD-细胞。首先门控CD3 +细胞，然后将该群体门控成CD4 +和CD4 -亚型。在CCR7和CD45RO标记的帮助下确定初始（CCR7 + CD45RO - ），中央记忆（CCR7 + CD45RO + ）和效应记忆（CCR7 - CD45RO + ; CCR7 - CD45RO - ））的比例。 <a href="http://ecsource.jove.com/files/ftp_upload/55591/55591fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55591/55591fig3.jpg" /> F图3:健康人眼表面的CD4 +和CD8 + T细胞中的免疫细胞亚型。在健康人类对照组中结膜CD4 +和CD8 +初始，中枢记忆（T CM ）和效应记忆（T EM和T EMRA ）亚群的分布;每个数据点表示单独的个体平均值±SEM。人口百分比在每个人口的名义下标记为红色。总人口的百分比为98％，因为CD4 + T EMRA群体不包括在上述散点图中（从Bose 等人 21修改）。 <a href="http://ecsource.jove.com/files/ftp_upload/55591/55591fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva at JoVE.com

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

暴露的正常眼表由角膜和结膜组成。上皮细胞，杯状细胞和免疫细胞存在于结膜中。这里，使用印模细胞学装置和流式细胞术来描述印象细胞学的非侵入性技术来分析结膜中的免疫细胞。

从结膜分离和表型细胞的非侵入性方式

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

a-non-invasive-way-to-isolate-and-phenotype-cells-from-the-conjunctiva

Research

JoVE Journal

Immunology and Infection

10.2K 次观看.  Nanyang Technological University. 暴露的正常眼表由角膜和结膜组成。上皮细胞，杯状细胞和免疫细胞存在于结膜中。这里，使用印模细胞学装置和流式细胞术来描述印象细胞学的非侵入性技术来分析结膜中的免疫细胞。 

视频: A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

遗传筛选隔离弓形虫宿主细胞的出口突变

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

非侵入性光学成像淋巴血管的鼠标

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

完整的胸腺切除术的成年大鼠非侵入气管插管

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...