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。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-human CD3 BV510</td><td>BD Biosciences</td><td>563109</td><td></td></tr><tr><td>anti-human CD4 APCH7</td><td>BD Biosciences</td><td>641398</td><td></td></tr><tr><td>anti-human CD45RO PECy7</td><td>BD Biosciences</td><td>337168</td><td></td></tr><tr><td>7-AAD solution</td><td>BD Biosciences</td><td>555816</td><td></td></tr><tr><td>anti-human CCR7 PE</td><td>BD Biosciences</td><td>552176</td><td></td></tr><tr><td>Pippetes</td><td>Eppendorf</td><td>NA</td><td></td></tr><tr><td>Local Anaesthesia</td><td>Alcaine</td><td>NA</td><td></td></tr><tr><td>Fluorescein sodium solution</td><td>Bausch &amp;amp; Lomb U.K Limited</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Keratograph 5M</td><td>Oculus</td><td>NA</td><td></td></tr><tr><td>Slit lamp BioMicroscope</td><td>Haag Streit</td><td>BM900</td><td></td></tr><tr><td>EyePrim</td><td>Opia Technologies</td><td>NA</td><td></td></tr><tr><td>FACS Verse</td><td>BD BioSciences</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GraphPad 6.0</td><td>Prism</td><td>NA</td><td></td></tr><tr><td>FACSVerse analysis software</td><td>BD</td><td>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

10.3K 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

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

科研

免疫与感染

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

生物工程

Impression Cytology of the Lid Wiper Area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

盖雨刮区的印象细胞学

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

医学

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

小鼠的共性共性隔离和识别

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

诱导眼表炎症和受累组织收集

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

分离、培养和鉴定人角膜缘生态位细胞的方案

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

作者聚焦:标准化角膜缘生态位细胞 （LNC） 分离和表征以支持广泛的 LNC 研究 

Here, we present a protocol to isolate and identify the human limbal niche cells.

角膜缘生态位细胞的分离和鉴定

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). In experimental animal models with certain genetic modifications or artificial operations, it is useful to examine all parts of the OS epithelial sheet to evaluate relative pathogenetic changes of each part in parallel. However, dissection of OS tissue as a whole without distortion or damage has been challenging, primarily due to the softness and thinness of the OS affixed to physically separate yet movable eyelids and eyeball. Additionally, the deep eye socket formed by the hard skull/orbital bones is fully occupied by the eyeball leaving limited space for operating dissections. As a result, direct dissection of the eyeball with associated OS tissues from the facial side would often lead to tissue damages, especially the palpebral and bulbar conjunctiva. In this protocol, we described a method to remove the skull and orbital bones sequentially from a bisected mouse head, leaving eyelids, ocular surface, lens and retina altogether in one piece. The integrity of the OS sheet was well preserved and could be examined by histology or immunostaining in a single section.

This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

分离完整眼珠以获得整体眼表面组织，用于组织学检查和免疫组织化学

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

生物学

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin that is secreted by conjunctival goblet cells (CGCs) onto the ocular surface contributes to multiple ocular surface diseases. The elimination of exogenous sex hormones is essential to obtain consistent results during in vitro study of sex-based differences in CGCs. This paper describes a method to minimize the presence of exogenous hormones in the study of sex-based differences in CGCs while maintaining their physiological function. CGCs from postmortem human donors of both sexes were cultured from pieces of the conjunctiva in RPMI medium with 10% fetal bovine serum (FBS) (referred to as the complete medium) until confluency. Nearly 48 h before the start of the experiments, CGCs were transferred to RPMI medium without phenol red or FBS but with 1% BSA (referred to as phenol-red-free medium). The normal cellular function was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) after carbachol (Cch, 1 x 10-4 M) stimulation using fura 2/acetoxymethyl (AM) microscopy. The result shows that CGCs maintained normal function in the phenol-red-free media after 48 h. No significant difference in [Ca2+]i response was observed between phenol red-free RPMI medium and complete medium upon Cch stimulation. Therefore, we recommend using the phenol-red free RPMI medium with 1% BSA to eliminate exogenous hormones without altering the normal function of CGCs in the study of sex-based differences.

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Phenol red-free/fetal bovine serum-free medium is a better option than advanced RPMI to eliminate exogenous hormones without altering the normal function of conjunctival goblet cells in the study of sex-based differences.

体外研究 研究结膜杯状细胞中基于性别的差异的方法

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin ...

从结膜分离和表型细胞的非侵入性方式

摘要

探索更多视频

从表型分析和人体皮肤淋巴细胞分离

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

盖雨刮区的印象细胞学

小鼠的共性共性隔离和识别

诱导眼表炎症和受累组织收集

角膜缘生态位细胞的分离和鉴定

角膜缘生态位细胞的分离和鉴定

角膜缘生态位细胞的分离和鉴定

分离完整眼珠以获得整体眼表面组织，用于组织学检查和免疫组织化学

体外研究研究结膜杯状细胞中基于性别的差异的方法

从结膜分离和表型细胞的非侵入性方式

摘要

探索更多视频

从表型分析和人体皮肤淋巴细胞分离

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

盖雨刮区的印象细胞学

小鼠的共性共性隔离和识别

诱导眼表炎症和受累组织收集

角膜缘生态位细胞的分离和鉴定

角膜缘生态位细胞的分离和鉴定

角膜缘生态位细胞的分离和鉴定

分离完整眼珠以获得整体眼表面组织，用于组织学检查和免疫组织化学

体外研究 研究结膜杯状细胞中基于性别的差异的方法

体外研究研究结膜杯状细胞中基于性别的差异的方法