We thank Dr. Tadashi Koyama for technical help. This work was supported in part by JSPS KAKENHI Grant Numbers 23249045, 15K09211, 15K19172; a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan; a grant for research on allergic disease and immunology, Japan.

1.准备肺上皮细胞的培养和<ol><li>培养A549人肺上皮细胞（贴壁细胞系）中的Dulbecco改进的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），100IU / ml青霉素，和100μg/ ml链霉素。 </li><li>卸下并丢弃从培养皿的细胞培养基，并用5洗一次 - 10毫升磷酸盐缓冲盐水（PBS）中。清洗后，立即吸出PBS。 </li><li>加2ml胰蛋白酶/乙二胺四乙酸（EDTA）（0.05％），在37℃下孵育，5％CO 2的3分钟。 </li><li>收集脱离的细胞在含有细胞培养基，离心在RT管在150×g下4分钟离心管中。 </li><li>悬浮细胞在2毫升细胞培养基的沉淀并除去对细胞计数的样品。算使用台盼蓝染色血球以检查细胞存活力细胞的数目。 </li><li>种子A549细胞上10厘米聚苯乙烯板在0.5的密度- 1.0×10 6细胞/皿含10毫升凝胶收缩测定培养基的。种子上6孔聚苯乙烯板的细胞以0.5的密度- 1.0×10 5个细胞/孔用2ml为EMT确认培养基。孵育细胞在37℃和5％CO 2的24小时。 </li></ol> 2. EMT过程<ol><li>添加TGF-β1（5微克/毫升）的10微升和10微升的TNF-α（10微克/毫升），以在步骤1.6接种的板凝胶收缩试验。添加TGF-β1（5微克/毫升）的2微升和2μl的TNF-α（10微克/毫升）至板在步骤1.6接种EMT确认。在37℃和5％CO 2的48小时。 </li></ol> 3.确认EMT过程的PCR和免疫印迹<ol><li>确认形态变化（由鹅卵石状，以纺锤形）用相差显微镜处理的细胞中。 注:规范人A549细胞有鹅卵石状和三角形的外观是上皮细胞的特征，但与TGF-β1和肿瘤坏死因子α刺激后，细胞出现长和梭形是类似于间充质细胞14,15。 </li><li>评估上皮标记物，如E-钙粘蛋白的表达，和间充质标记物如N-钙粘蛋白，波形蛋白和α平滑肌肌动蛋白使用PCR或印迹。 <ol><li>使用RNA提取试剂盒16中的细胞中提取总RNA，并使用根据制造商的协议逆转录17合成cDNA。 </li><li>测量使用实时PCR系统18，并根据制造商的说明单体花青染料PCR试 ​​剂盒的mRNA水平的表达。对于GAPDH特异性引物（甘油醛-3-磷酸脱氢酶），ACTA2（的α-肌动蛋白2），CDH1（钙粘蛋白-1; E-钙粘蛋白），和VIM（波形蛋白）示于表1中 </st荣&gt;。 </li><li>裂解用裂解缓冲液（ 表2）含有1％蛋白酶抑制剂混合物溶液中的细胞。测量使用蛋白测定试剂盒19,20的所有样品的蛋白浓度，并应用相同量的蛋白质，以聚丙烯酰胺凝胶。 <ol><li>执行SDS凝胶电泳和蛋白质的半干转移到PVDF膜21。 2小时 - 在1封闭缓冲液孵育初级抗体的膜。温育后，用洗涤缓冲液洗两次膜孵育与第二抗体在膜1小时。 注:请参阅材料/设备表在这些研究中使用的抗体和稀释。 见表2为封闭缓冲液的组分。 </li><li>两次洗涤用洗涤缓冲液的膜。就拿膜的照片与使用冷CCD相机11,23蛋白质印迹检测试剂盒22。 </li></ol></li></ol></li></ol> 4。凝胶收缩试验评定EMT <ol><li>吸从细胞培养容器中的条件培养基，并用5洗井 - 10mL的PBS，以去除死细胞。清洗后，立即吸出PBS。 </li><li>加2ml的0.05％胰蛋白酶/ EDTA中，并在37℃反应和5％的CO 2 3分钟。 </li><li>收集脱离的细胞在含有DMEM补充有蛋白酶抑制剂（1毫克/毫升），离心管在室温下在150×g下4分钟离心管中。 </li><li>混合型胶原凝胶用蒸馏水，和4×浓缩的DMEM以调节体积以达到1.75毫克/毫升的胶原浓度，和1×DMEM的浓度。一定要保持冰凝胶培养基在这一步。 注意:为了让6毫升凝胶培养基中，拌匀3.5毫升1型胶原凝胶（3毫克/毫升），1.5毫升4×浓的DMEM和1 ml蒸馏水。 </li><li>悬浮于500μlPBS中的细胞沉淀并除去对细胞计数的样品。计数使用血球用锥虫蓝染色的细胞的数目来检查细胞存活力。 </li><li>添加凝胶介质来调整卷来实现/孔3.0×10 5个细胞的细胞密度（6.0×10 5个细胞/ ml），轻轻地但很快没有凝胶化，通过移液混合。 </li><li>分配将0.5ml混合物成24孔未处理的板的每个孔迅速并小心地使整齐圆筒形。要小心，不要让任何气泡污染凝胶（ 图1A）。 注意:含有细胞的凝胶介质是粘性的，并且可以很容易地在井胶凝并形成月牙形状。 </li><li>在37℃下孵育在细胞培养箱15分钟的盘，5％CO 2和95％的湿度完全胶凝。 </li><li>分离从板凝胶而不通过的方式移动的无菌刮刀在一个方向上绘制的圆周断裂。用刮刀轻轻转移凝胶至60mm组织培养皿含有5毫升含或不含TGF-β1（5毫微克/毫升）和TNF-α（10毫微克/毫升）的DMEM / 1％FBS的。 </li><li>轻轻摇动菜，以确保凝胶漂浮在介质上。在37℃，5％CO 2和95％湿度下孵育在细胞培养箱中。 </li></ol> 5.凝胶尺寸的测量<ol><li>后使用图象分析系统0，24，48，和72小时测量胶原蛋白凝胶的尺寸。 <ol><li>打开凝胶文档系统（ 图2A），并把餐具放入光屏蔽柜。然后起飞的内阁菜盖子。 </li><li>打开相关的凝胶分析软件。单击菜单栏中的"图像采集按钮"，显示在机柜凝胶的形象。然后，点击"获取"，在菜单栏取凝胶（ 图2B）的照片。 </li><li>单击菜单栏（ 图2C）中的"检测键"，调整测量区域（黄圈我Q 图2C）通过拖动鼠标。然后，单击菜单栏中的"自动检测按钮"（ 见图2D按钮）。该软件会自动检测出热图（ 图2D）凝胶。 </li><li>点击"确定"，以提取图像处理凝胶的轮廓和计算的轮廓（ 图2E）所包围的区域。的区域被计算后，将计算出的区域被显示在单独的弹出窗口。 注意:如果凝胶成像期间彼此重叠，轻轻用无菌移液管尖移到凝胶。 </li></ol></li></ol>

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作者有没有利益冲突披露。

在这项研究中开发的协议包括两个步骤。进行第一步骤以诱导EMT的，而第二个步骤是在凝胶收缩试验。因为它确认细胞发生EMT是很重要的，第2步提供了一个极好的补充，形态学和基因表达的变化。以往的研究表明，A549细胞的EMT诱导仅24，TGF-β1;然而，正如我们以前10日报道，TNF-α处理增强EMT和收购的间充质细胞标志物。它被认为是TGF-β介导EMT的机制是SMAD依赖性25。 TNFα增强了TGF-β1诱导的EMT，导致增强凝胶收缩。已经报道了TNFα的用于增强TGF-β1诱导的EMT的机制不仅Smad2的接头区的磷酸化，但也MAPK信号调节26。因此，在该研究开发的协议包括stimul由两个TGF-β1和TNF-α的通货膨胀。 该行的EMT到I型胶原凝胶的类型，和浮动在介质上的细胞的嵌入，是该测定（步骤4）的中央的方面。由于凝胶是软的，容易损坏，并且在每侧有不同的尺寸，施加凝胶时向培养基中并测量它们的尺寸格外小心是必需的。如果难以进行这种步骤中，是测量井的凝胶尺寸而不凝胶移入60mm培养皿27,28的替代方法。有几个共同的问题，第一个问题是，胶原凝胶容易凝胶化在RT，所以凝胶如在步骤4.7中提到经常变形。因此，该板块一定要轻轻地铸造成凝胶井，以确保他们采取一个整洁的圆柱状后动摇。第二个问题是，如果任何气泡凝胶化期间输入的凝胶，然后将凝胶的大小将是不均匀的。要小心，不要让任何空气bubbl下载到污染凝胶。有原始的方法，这方法之间有两个主要差异。第一差别是该原始方法的胶原凝胶的最终浓度为0.75毫克/毫升，但是，该方法的是1.75毫克/毫升，以使凝胶收缩超过他们做的原始方法。第二差异是，原来的方法使用凝胶浮动平台无血清培养基，但这种方法中，以保持细胞生存力和维持细胞收缩使用1％FBS中的凝胶的浮动平台。 最初为成纤维细胞开发的凝胶收缩试验是在体外模型用于评估有助于伤口愈合和纤维化13的过程的细胞的收缩性的理想。成纤维细胞的1型胶原的连接应该是产生机械张力该因而导致在胶原凝胶的尺寸的减小。此外，该测定中，最近已经使用作为研究的炎性疾病，包括支气管哮喘和COPD 29-31气道细胞的收缩的体外模型。 凝胶收缩试验是可比其他测定法，诸如细胞迁移测定的，因为它不需要特定设备和显示出高的定量重复性。但是，也有施加凝胶收缩试验用于评估EMT 32报道很少。在这项研究中，包含该行的EMT的细胞凝胶显著在大小减小24到72小时的凝胶化后，指示细胞收缩。然而有这种方法的局限性。第一个是，在该测定中，凝胶的尺寸变化显示在凝胶所有细胞的收缩力的总和。然而，这是难以评价在使用该测定的凝胶单细胞收缩。第二个是，A549细胞是癌症细胞系，而不是正常的人上皮细胞。因此，它是难以推断ö乌尔结果直接肺纤维化的发病机制。然而，A549细胞被广泛用于研究呼吸道上皮细胞的功能，并且一些研究通过使用A549细胞肺纤维化33,34的模型得到的EMT的证据。 总之，I型胶原蛋白包含经历EMT的大小减少了，指示这些细胞的收缩间充质细胞的凝胶。因此，凝胶收缩试验应被视为一个扩展体外测定中通过EMT过程中的细胞，以评估采集收缩功能。

纤维化参与多种慢性进行性疾病，如间质性肺疾病，心脏纤维化，肝硬化，终端肾功能衰竭，系统性硬化，和自身免疫性疾病1的发病机制。间间质性肺疾病，特发性肺纤维化（IPF）是一种慢性进行性疾病，并显示预后差。 IPF的病理特点是成纤维细胞灶组成了与预后有关激活成纤维细胞和肌的发展。提出这样的肺成纤维细胞的起源是从几个间充质细胞，包括原驻地肺成纤维细胞和骨髓循环纤维细胞衍生的。最近，上皮-间质转化（EMT）已经提出了用间充质细胞2的形成相关联，并且有助于纤维化疾病的发病机制。 它被认为是电磁ŧ在胚胎发育过程中的重要作用，伤口愈合和癌症的进展，包括肿瘤侵袭和转移3。以下EMT的过程中，上皮细胞的上皮标记物，如E-钙粘蛋白的损失获得间充质细胞的能力，以及由间充质标志物，如波形蛋白和α平滑肌肌动蛋白（SMA）4,5的表达。以往的研究表明，EMT过程已与组织纤维化的发展在肾脏6和肺7被相关联的证据。此外，慢性炎症促进纤维化疾病8;此外，如炎性细胞因子如肿瘤坏死因子超家族成员14（TNFSF14; LIGHT），肿瘤坏死因子（TNF）-α和白细胞介素1β，已显示出提高的EMT 9-12。 胶原凝胶收缩试验，其中成纤维细胞嵌入型我一个基于胶原的细胞收缩测定胶原凝胶三维， 在体外模型收缩的评价理想。收缩是成纤维细胞的特征功能之一，并有助于正常的伤口修复和纤维化13。在该测定中，可以认为，成纤维细胞附着于I型通过整合素依赖性机制胶原被认为在一定条件下，以产生机械张力，并因此导致组织收缩。 这里，凝胶收缩测定的发展报告可以适于评估在经历EMT的细胞中采集收缩功能。这份报告表明，这种改进法是适用于评估在经历了EMT间质细胞收缩。

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:167px;">For A549 medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:96px;">GIBCO</td>
			<td style="width:119px;">10437</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Transforming Growth Factor-&#946;1, Human, recombinant</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">209-16544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinant Human TNF-&#945;</td>
			<td style="width:96px;">R&#38;D systems</td>
			<td style="width:119px;">210-TA/CF</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">E-Cadherin (24E10) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#3195</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vimentin (D21H3) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#5741</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-&#945;-Tubulin antibody</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">T9026</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-Actin, &#945;-Smooth Muscle antibody&#160;</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">A5228</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-N-cadherin antibody</td>
			<td style="width:96px;">BD Transduction Laboratories</td>
			<td style="width:119px;">#610920</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA931-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">blocking reagent</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN418</td>
			<td>2% in TBS-T</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid</td>
			<td style="width:96px;">corning</td>
			<td style="width:119px;">#353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mm cell culture dish</td>
			<td style="width:96px;">TPP</td>
			<td style="width:119px;">#93100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, powder</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">12100-046</td>
			<td>For 4&#215;DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">type 1 collagen gel</td>
			<td style="width:96px;">Nitta gelatin</td>
			<td style="width:119px;">Cellmatrix type I-A</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">24 well cell culture plate</td>
			<td style="width:96px;">AGC TECHNO GLASS</td>
			<td style="width:119px;">1820-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel Documentation System&#160;</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">AE-6911FXN</td>
			<td>Gel imager</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">gel analyzing software</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">Densitograph, ver. 3.00</td>
			<td>analysing software bundled with AE-6911FXN</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 Well Plates, Non-Treated</td>
			<td style="width:96px;">IWAKI</td>
			<td style="width:119px;">1820-024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNA extraction kit</td>
			<td style="width:96px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">reverse transcriptase</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">18080044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">real time PCR system</td>
			<td style="width:96px;">Stratagene</td>
			<td style="width:119px;">Mx-3000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR green PCR kit</td>
			<td style="width:96px;">Qiagen</td>
			<td style="width:119px;">204145</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease Inhibitor Cocktail (100X)</td>
			<td style="width:96px;">life technologies</td>
			<td style="width:119px;">78429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVDF membrane</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">2392390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">protein assay kit</td>
			<td style="width:96px;">bio-rad</td>
			<td style="width:119px;">5000006JA&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyacrylamide gel</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">2331810</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">western blotting detection reagent</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN2232</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cold CCD camera</td>
			<td style="width:96px;">ATTO</td>
			<td style="width:119px;">Ez-Capture MG/ST</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin inhibitor</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">T9003-100MG</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Polyoxyethylene (20)Sorbitan Monolaurate</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">163-11512</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">polyoxyethylene (9) octyiphenyl ether</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">141-08321</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an in vitro assay to evaluate contractile function of mesenchymal cells that underwent epithelial-mesenchymal transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

EMT期间，上皮细胞失去上皮标记，如E-钙粘蛋白，并获得间充质标志物，如波形蛋白和α平滑肌肌动蛋白4,5的表达。与TGF-β1和肿瘤坏死因子αA549人肺上皮细胞的温育诱导EMT。正常A549细胞的外观是鹅卵石状的形状，三角形形状是上皮细胞（ 图3A）的特性，但是刺激的TGF-β1和TNF-α后，外观改变为长纺锤形是类似于间充质细胞（ 图3B）。 的上皮细胞和间质标记物的表达进行评价，确认细胞发生EMT。相对mRNA的表达与ΔΔCT法计算。个人数据被归一针对甘油醛-3-磷酸脱氢酶（GAPDH），它是一个内务克烯。与TGF-β1和肿瘤坏死因子α48小时处理的A549细胞已显著减少CDH1的表达和显著增加VIM和ACTA2（ 图4A）的表达。用于Q-PCR的引物序列示于表1。如在图4B中 ，刺激TGF-β1和肿瘤坏死因子α减毒E-钙粘蛋白的表达示出，而波形蛋白，N-钙粘蛋白，α平滑肌肌动蛋白的表达诱导。 进行凝胶收缩试验，以评估经历EMT的细胞的收缩性。诱导与TGF-β1和肿瘤坏死因子αEMT后，将细胞浇铸成胶原蛋白凝胶并孵育含有TGF-β1和TNF-α，或PBS 72小时中的介质。 如图5A所示，含有TGF-β1和TNF-α处理的细胞中的凝胶比含有用PBS处理的细胞控制凝胶小。要QUAntify在凝胶大小的变化，凝胶使用凝胶分析器0，24，48，和处理后72小时进行分析。 72小时后，含有TGF-β1和肿瘤坏死因子α处理的细胞凝胶的尺寸均显著减少与对照相比的凝胶（ 图5B）。我们还证实，含有TGF-β1处理的细胞的凝胶单独比对照凝胶小，但为（数据未显示）比TGF-β1和肿瘤坏死因子α处理的凝胶不小。 <img alt="图1" src="/files/ftp_upload/53974/53974fig1.jpg" /> 图1.补充图的制作凝胶。这些图像显示抛入井中的凝胶。含有细胞的凝胶介质是粘性的，并且可以容易胶凝并在井形式月牙形。 （A）左边的图像显示，该凝胶被正确铸造，右边的图片显示"整洁的圆筒形"的模式。 （B &lt;/强&gt;）左边的图像显示，该凝胶变形，并且右图像显示，气泡污染凝胶。 （C）的凝胶是软的，这样容易损坏，变形，通常像"吃豆人"， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53974/53974fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53974/53974fig2.jpg" /> 图2中的步骤测量的凝胶尺寸（A）的凝胶文档系统包括PC和凝胶成像系统。 （B）中的"图像获取按钮"，和凝胶的照片。 （C）中的"检测键"，和用于调整测量区域（黄色圆圈）的窗口。 （D）的"自动检测键"，并且该软件使得凝胶的热图从一个图象到脱<html> TECT凝胶的轮廓。 （E）该软件通过提取图像处理凝胶的轮廓，并计算出轮廓包围的地区。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53974/53974fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53974/53974fig3.jpg" /> 图3. EMT诱导细胞形貌。A549人肺上皮细胞以1.0×10 5个细胞/孔的密度在一个6孔板中铺板并有或没有5纳克/ ml的TGF-β1和10ng / ml的温育TNF-α为48小时，接着通过相差显微成像。 （A）和（B）是下×200所获得的图像。在（A）的比例尺和（B），100微米。<html> T ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53974/53974fig4.jpg" /> 图4. EMT刺激与TGF-β1和肿瘤坏死因子α。 （A）EMT标志物的表达（CDH1，VIM和 ACTA2）的定量RT-PCR分析。 （B）中示出E-钙粘蛋白，N-钙粘蛋白，波形蛋白的表达Western印迹，和α平滑肌肌动蛋白在有或无TGF-β1和TNF-α刺激的A549细胞的蛋白质。 α微管蛋白用作内部对照。 A549细胞分别为在图1中，N = 3次独立实验中培养。 ** P &lt;0.01;误差棒代表SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53974/53974fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 尔斯/ ftp_upload / 53974 / 53974fig5.jpg"/&gt; 图5.细胞发生EMT获得收缩 。在10 cm的培养皿1.0×10 6个细胞/皿的密度铺板并有或没有5纳克/ ml的TGF-β1和10ng / ml的TNF-α的48小时培养A549细胞。然后将细胞在24孔板铸造成500微升含有1.75毫克/毫升的胶原凝胶培养基以3.0×10 5个细胞/孔的密度。后的凝胶形成，它们被添加到培养基有或没有5纳克/ ml的TGF-β1和10ng / ml的60毫米培养皿中TNF-α和培养72小时，随后通过成像（A）中。凝胶尺寸被后使用图像分析系统0，24，48，和72小时进行测定。 N = 9个独立的实验（B）中。 ** P &lt;0.01;误差棒代表SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53974/53974fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:kEEP-together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 基因名称 </td><td colspan="2"> 前进5&#39;→3&#39; </td><td colspan="2"> 扭转5&#39;→3&#39; </td></tr><tr><td> ACTA2 </td><td colspan="2"> GCACCCAGCACCATGAAGA </td><td colspan="2"> ACCGATCCAGACAGAGTATTT </td></tr><tr><td> GAPDH </td><td colspan="2"> GGTGAAGGTCGGAGTCAACGGA </td><td colspan="2"> GAGGGATCTCGCTCCTGGAAGA </td></tr><tr><td> VIM </td><td colspan="2"> GACAATGCGTCTCTGGCACGTCTT </td><td colspan="2"> TTCTTCTGCCTCCTGCAGGTTCTT </td></tr><tr><td> CDH1 </td><td colspan="2"> CCCATCAGCTGCCCAGAAAATGAA </td><td colspan="2"> CTGTCACCTTCAGCCATCCTGTTT </td></tr></tbody></table> 表1引物序列。用于定量RT-PCR引物序列。 GAPDH用作内部对照。 <table border="1" fo:keep-together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 裂解液:RIPA缓冲液 </td><td></td></tr><tr><td>的20mM的Tris-HCl pH为7.5 </td><td></td></tr><tr><td> 150毫米氯化钠</td><td></td></tr><tr><td> 1毫摩尔EDTA </td><td></td></tr><tr><td> 1％聚氧乙烯（9）醚octyiphenyl </td><td></td></tr><tr><td> 0.1％的钠脱氧胆酸</td><td></td></tr><tr><td> 0.1％SDS中</td><td></td></tr><tr><td> 封闭液 </td><td></td></tr><tr><td>在洗涤缓冲液的1％封闭试剂</td><td></td></tr><tr><td>洗涤液:TBST缓冲液</td><td></td></tr><tr><td>氯化钠</td><td> 45克</td></tr><tr><td>的1M Tris pH 7.4的</td><td> 50毫升</td></tr><tr><td>聚氧乙烯（20）脱水山梨醇单月桂酸</td><td> 2.5毫升</td></tr><tr><td>水蒸馏</td><td>加入5升</td></tr><tr><td&gt;总</td><td> 5升</td></tr></tbody></table> 表2缓冲器的使用的组分。裂解的组分，封闭缓冲液和洗涤缓冲液。

Watch this Scientific Journal Video about Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition at JoVE.com

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

development-an-vitro-assay-to-evaluate-contractile-function

Nanyang Technological University

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JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

12.1K 次观看.  The University of Tokyo Hospital. Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

视频: Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

科研

发育生物学

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...