这项工作得到了韩国国家研究基金会 (2015R1D1A1A01060839) 和韩国政府 (NRF) 资助的韩国国家研究基金会 (MSIT) 的资助 (不 2016R1A2B2008887, 不。2016R1A5A2007009) (Jeanho 云)。

1. 诱发癌基因引起的衰老
<ol><li>
制备 RasV12 逆转录病毒
	<ol>
<li>在室温下添加2毫升0.001% 聚 l-赖氨酸/磷酸盐缓冲盐水 (PBS), 将 100 mm 培养皿涂成5分钟。</li>
		<li>使用连接到真空的玻璃吸管去除聚 l-赖氨酸溶液, 并通过添加2毫升 1x PBS 来洗涤培养皿。</li>
		<li>板 3 x 106 ecotropic BOSC-23 包装细胞在涂层培养皿与 Dulbecco 的改性鹰培养基 (DMEM) 含有10% 胎牛血清 (血清) 和1% 青霉素/链霉素, 并培养他们在37°c 的共同2组织培养孵化器 1 d。</li>
		<li>第二天, 染2µg 的 pBabe puro-H-RasV12 dna 与逆转录病毒包装 dna (2 µg 的 pGAG/波兰和0.25 µg pVSVG) 使用一个转染试剂 8 H 根据制造商的指示。</li>
		<li>去除转染介质, 加入8毫升的新鲜介质。</li>
		<li>48小时后, 收集含有病毒粒子的介质, 并通过离心 500 x g以5分钟的方法去除任何细胞碎片。</li>
		<li>用0.45 µm 注射器过滤器过滤含 h-Ras 病毒的介质。 注意: 避免任何冰冻和解冻的病毒上清液有效的病毒感染。要获得最佳效率, 请使用在感染的同一天收获的病毒。</li>
	</ol>
</li>
	<li>
H-RasV12 逆转录病毒感染 WI-38 正常人成纤维细胞
	<ol>
<li>板 5 x 104 WI-38 细胞在60毫米培养皿与 DMEM 包含10% 个血清和1% 青霉素/链霉素 1 d 在收获 H-RasV12 逆转录病毒之前, 并且培养他们1天在37°c。</li>
		<li>第二天去除生长培养基, 加入1毫升的 H-RasV12 逆转录病毒培养基。添加额外的1毫升的生长培养基, 含有2µL 的凝聚胺溶液 (8 毫克/毫升蒸馏水)。在湿润37°c, 5% CO2组织培养孵化器中孵化样品1天。</li>
		<li>去除 H-RasV12 逆转录病毒混合物24小时后, 用 1x PBS 清洗细胞2x。添加生长培养基和治疗细胞与2µg/毫升嘌呤霉素2天的湿润37°c, 5% CO2组织培养孵化器。</li>
		<li>经过2嘌呤霉素选择, 去除生长培养基, 并用 1x PBS 清洗细胞2x。添加生长培养基和培养细胞在一个湿润的37°c, 5% CO2孵化器, 直到指定的时间点 (见图 1A的示意图)。</li>
	</ol>
</li>
</ol>2.通过衰老相关的β-乳糖酶染色监测衰老
<ol><li>
准备以下库存解决方案。
	<ol>
<li>在二甲酰胺 (DMF) 中制备20毫克/毫升 5-溴-4-氯-3-哚β-galactopyranoside (X 加仑)。将解决方案存储在摄氏-20 摄氏度。</li>
		<li>在126毫升蒸馏水中溶解3.377 克钠2HPO4. 7H2O (0.708 毫米) 和36.8 克柠檬酸 (100 毫米), 制备柠檬酸/磷酸钠缓冲液。如有必要, 将 pH 值调整为6.0。</li>
		<li>准备0.5 米盐钾。在4摄氏度的黑暗中存储解决方案。</li>
		<li>准备0.5 米氰化钾钾。在4摄氏度的黑暗中存储解决方案。 注: 与衰老相关的β-乳糖酶 (SA β-加仑) 染色溶液的 pH 值对于准确的结果是至关重要的。</li>
	</ol>
</li>
	<li>通过稀释含有150毫米氯化钠、2毫米氯化镁2、5毫米钾盐、5毫米钾氰化钾、20% (v/v) 柠檬酸/磷酸钠缓冲液和1毫克/毫升 X 加仑的库存溶液, 制作新鲜的 SA β-gal 染色液。</li>
	<li>卸下培养基并用 1x PBS 清洗单元格1x。室温下用3% 甲醛固定细胞5分钟。 注意: 未感染逆转录病毒的细胞应用作阴性对照。未感染的控制细胞应继续传代以保持其增殖状态。</li>
	<li>从细胞中取出固定液, 用 1x PBS 冲洗。添加新鲜准备的 1x SA β-gal 染色溶液 (1.5 毫升在60毫米培养皿)。用石蜡膜封住盘子以防止干燥, 在37摄氏度时将其孵化24小时。或者, 在湿度室中一起孵化盘子。 注: 含有3% 甲醛的固定液应作为危险废物处置。没有 CO2的孵化器是可取的, 以防止在孵化期间 pH 值的变化。</li>
	<li>第二天检查显微镜下细胞的染色状态;在核周地区, SA β-gal 阳性细胞呈蓝色。 注: 增殖控制细胞显示低频率的 SA β-gal 阳性细胞。如果染色太轻, 继续孵化一段较长的时间 (见图 1B的代表性例子)。</li>
	<li>使用10X 或更大物镜获取带有 CCD 摄像机的 SA β-gal 染色图像。捕获足够数量的图像来计算染色百分比 (> 200 细胞)。</li>
	<li>通过量化 sa β-gal 阳性细胞的数量相对于单元格总数, 计算 sa β-gal 染色细胞的百分比。 注: SA β-gal 染色应单独重复至少 3x, 并应根据复制实验结果计算平均值 (图 1C)。适当的细胞密度对 SA β-gal 染色实验至关重要。高融合可以干扰衰老相关形态学的改变, 并在控制细胞中诱发假阳性信号。</li>
</ol>3. 在 H-Ras 诱导衰老过程中量化活性氧的诱导
<ol><li>板 2 x 105 WI-38 细胞在60毫米培养皿和挑衅 H Ras 导致的衰老如步骤1所述。 注: 本协议可应用于其他类型的细胞衰老模型。</li>
	<li>在指定的时间点 (2、4或6天) 移除生长介质, 并用 1x PBS 冲洗细胞。用1毫升0.05% 胰蛋白酶/EDTA 溶液将细胞分离, 并通过增加1毫升的生长培养基来使胰蛋白酶钝化, 其中含有10% 的血清。确定单元格计数。</li>
	<li>将 1 x 105细胞转移到15毫升圆锥管中, 并通过离心在 500 x g处收集细胞以5分钟。</li>
	<li>制备新鲜的 2 ', 7 '-dichlorofluorescin 双乙酸 (dcf-da) 染色介质通过增加50µL 10 毫米 DCF-da 到10毫升培养基 (dcf 的工作浓度为50µM)。 注: DCF-DA 是轻敏感的。应避免轻曝光。根据细胞类型的不同, 可以调整 DCF 浓度和染色时间。</li>
	<li>从15毫升锥形管中仔细去除生长介质, 加入1毫升新鲜制备的 DCF-DA 染色培养基。仔细并用重悬细胞颗粒, 在黑暗中孵化37摄氏度, 30 分钟。包括无染色样品作为阴性染色控制。 注: 未沾上 DCF 的增殖细胞应作阴性对照。</li>
	<li>通过离心 500 x g 5 分钟收集细胞, 并用2毫升 1x PBS 清洗1x。并用重悬1毫升 1x PBS 的细胞, 并将样品转移到适当的荧光活化细胞分选管。</li>
	<li>
使用配备适当激光源的流式细胞仪测量-2′-、7′-Dichlorodihydrofluorescein 双乙酸 (DCF) 荧光信号。用蓝色激光 (488 nm) 激发样品, 用对数刻度检测出 530 # 30 nm 探测器 (FITC) 发出的荧光。
	<ol>
<li>首先分析非染色阴性控制单元。在一个点图的正散射 (FSC)与侧面散射 (SSC), 选择活细胞使用浇口工具, 并消除死细胞和细胞碎片。 注意: 在 FSC与SSC 的点图中应该仔细监视细胞大小的变化。如果衰老细胞的大小显著增加, 则在 FSC与SSC 的点图中, 在相同大小的细胞群中, 应将 DCF 的强度进行比较。</li>
		<li>在单参数直方图中, 在x轴的对数刻度上显示 DCF (488 nm) 荧光, 以及y轴线性刻度上的事件数 (单元数), 调整 488 nm 激光器的电压, 将峰值从左边缘的非染色细胞。</li>
		<li>分析每样样品的流量, 并记录至少1万件事件。使用特定于流式细胞仪的分析软件获取每个样本的平均荧光值。 注: ROS 测量应该独立地重复至少 3x, 并且平均值应该从这些结果计算。典型的 H-Ras 引起的衰老结果如图 2所示。</li>
	</ol>
</li>
</ol>4. 利用实时聚合酶链反应量化 IL-6 和 IL-8 mRNA 表达与衰老相关的分泌表型分析
<ol><li>
总 RNA 准备
	<ol>
<li>板 3 x 105 WI-38 细胞在100毫米培养皿在 DMEM 包含10% 个血清和1% 青霉素或链霉素, 并且培养他们在37°c 在一个组织培养孵化器为1天。为每个时间点准备重复的区域性。</li>
		<li>通过在步骤1中所述的 H-RasV12 逆转录病毒感染细胞来诱导衰老。</li>
		<li>在收割前1天, 用 1x PBS 洗涤细胞 2x, 并添加3毫升的 DMEM, 不需加血清。 注: 这一步骤是为了生产用于 ELISA 的条件介质, 如步骤5所述。血清饥饿可诱发 IL-6 和 IL-8 基因在某些敏感细胞中的表达。因此, 应首先比较 IL-6 和无血清饥饿培养细胞中的 IL-8 mRNA 表达。如果血清饥饿引起 IL-6 或 IL-8 mRNA 表达的显著变化, 则应分别制备 qPCR 的总 RNA 和 ELISA 的条件培养基。</li>
		<li>收集每样样品24小时后的条件培养基, 并存储培养基在-80 °c 的 ELISA。</li>
		<li>用1毫升0.05% 胰蛋白酶/EDTA 溶液将细胞分离, 并通过增加1毫升的生长培养基来使胰蛋白酶钝化, 其中含有10% 的血清。确定 ELISA 结果规范化的单元计数, 如步骤5所述。以 500 x g为5分钟, 在1.5 毫升离心管中采集细胞。</li>
		<li>用温和的吸力去除培养基, 加入1毫升的 RNA 萃取液。然后, 离心管在十五年代大力涡旋。随后, 增加200µL 氯仿和强烈漩涡混合物再为另外的十五年代。</li>
		<li>离心样品管在 1.7万 x g 10 分钟在4°c。然后, 小心地将上相转移到一个新的1.5 毫升离心管。 注: 在上部相转移到新管时, 界面和下部有机相不应受到干扰。</li>
		<li>添加400µL 的异丙醇沉淀 RNA, 并通过温和的反转混合。然后, 在室温下孵化管10分钟。</li>
		<li>离心管在 1.7万 x g 10 分钟在4摄氏度。然后, 丢弃上清, 注意保留 RNA 颗粒。通过添加1毫升75% 乙醇和反转管2–3x 来洗涤 RNA。离心管为5分钟在 1.7万 x g在4°c 和放弃上清。</li>
		<li>在室温下干燥 rna 颗粒, 在焦碳酸 (DEPC) 处理的蒸馏水中溶解 rna 50 µL。利用1µL rna 溶液, 用分光光度法定量测定总 rna 浓度。 注: Overdrying 可降低 RNA 的溶解度;因此, 避免 overdrying RNA。</li>
	</ol>
</li>
	<li>
cDNA 制备
	<ol>
<li>将以下内容添加到薄壁 PCR 管中, 为每个样品准备反向转录反应混合物: 2 µg 总 RNA (调整容量到10µL 以 RNase 无水), 2 µL 10x RT 缓冲器, 1 µL 一个随机底漆 (50 pmol), 0.8 µL 25x dNTP 混合 (100 毫米), 1 µL MMLV 逆转录酶 (50 U/µL), 5.2 µL 无 RNase 水。</li>
		<li>在热循环仪上设置反向转录反应程序, 条件如下:42 °c 为60分钟, 95 摄氏度为5分钟. 将 PCR 管放在热循环仪中, 启动程序。</li>
	</ol>
</li>
	<li>
实时聚合酶链反应
	<ol>
<li>为所有反应准备实时 PCR 大师组合。每个样品的反应混合物如下:25 µL 的2x 实时 PCR 总组合, 1 µL 每一个向前和反向底漆 IL-6 或 IL-8, 21 µL 的超纯净蒸馏水 (DNase 和 RNase)。见表 1。 注: 为每个含有肌动蛋白 RNA 引物的样品准备实时 PCR 反应混合物作为内部控制。GAPDH 还可以用作规范化的内部控制。</li>
		<li>在96井光学 qPCR 板中添加48µL 的主混合和2µL 的3倍稀释 cDNA 产品与水。每三个反应进行三份。准备一个不包含 cDNA 模板作为 PCR 控制的控制井。</li>
		<li>在热循环仪上建立实时 PCR 反应程序, 条件如下:10 分钟在95°c, 40 循环 of15 s 在95°c (变性), 并且1分钟在60°c (退火和引伸)。</li>
		<li>在 pcr 循环完成后进行实时 pcr 和记录阈值周期 (CT)。使用 2-ΔΔCΤ方法20计算 IL-6 或 IL-8 的 mRNA 水平。 注意: 表达式中的相对褶皱变化是在规范化到肌动蛋白水平之后, 使用以下公式确定的: 折变 = 2-Δ (ΔCT) 这里 ΔC , IL-6 或 IL-8t,肌动蛋白;和 Δ (ΔCt) = ΔCt, 被刺激的ΔCt,控制.
</li>
		<li>计算每个重复样本的平均值。 注: qPCR 应独立地重复至少 3x, 并根据这些结果计算平均值。典型的 H-Ras 引起的衰老结果如图 3所示。血清饥饿可诱发 IL-6 和 IL-8 基因在某些敏感细胞中的表达。因此, 应首先比较 IL-6 和无血清饥饿培养细胞中的 IL-8 mRNA 表达。如果血清饥饿引起 IL-6 或 IL-8 mRNA 表达的显著变化, 则应分别制备 qPCR 的总 RNA 和 ELISA 的条件培养基。</li>
	</ol>
</li>
</ol>5. 用 ELISA 方法定量测定与衰老相关的分泌表型分析中分泌的 IL-6 和 IL-8 蛋白的含量
<ol><li>解冻3毫升的条件培养基收获从衰老 WI-38 细胞如步骤4所述。以 500 x g为5分钟的离心去除细胞碎片。</li>
	<li>用离心过滤装置将条件介质10倍的离心浓缩为 3000 x克, 以20分钟为单位。 注意: 条件介质的浓度不一定取决于样品类型。</li>
	<li>
用适当的 IL-6 和 IL-8 elisa 试剂盒, 使用每个浓缩样品的50µL 进行 elisa。 注: 在两个 ELISA 井中测试每个样本。由于每个时间点在步骤4中都重复, 因此此分析允许我们监测 ELISA 检测和样品制备技术的变化。<ol>
<li>对于 ELISA 板涂层, 稀释 IL-6 或 IL-8 抗体与 1x PBS 的浓度为0.5 µg/毫升为 IL-6 或0.125 µg/毫升 IL-8。添加100µL 的稀释抗体的每一个良好的 ELISA 板。用 ELISA 钢板密封膜密封钢板, 在室温下进行孵化, 不需夜间晃动。</li>
		<li>通过吸入去除抗体溶液。洗净每个井4x 与300µL 1x 洗涤缓冲器 (0.05% Tween-20 在 PBS)。向每个井中添加300µL 的阻塞缓冲器 (PBS 中的 1% BSA)。室温孵育1小时。</li>
		<li>通过吸尘消除阻塞解决方案。用300µL 1x 洗涤缓冲器清洗每井4x。在稀释缓冲器 (PBS 中的 0.05% Tween-20 和 0.1% BSA) 中制备连续稀释的 ELISA 标准, 以生成标准曲线。 注: IL-6 的系列稀释为31.25、62.5、125、250、500、1000和 2000 pg/毫升。IL-8 的系列稀释为2.34、4.69、9.38、18.75、37.5、75和 150 pg/毫升。</li>
		<li>添加100µL 的标准溶液或100µL 的样品溶液 (50 µL 的浓缩介质与50µL 稀释缓冲), 每井。在室温下孵化水井2小时。</li>
		<li>取出标准或样品解决方案的愿望。用300µL 1x 洗涤缓冲器清洗每井4x。稀释的检测抗体与稀释缓冲液浓度为0.1 µg/毫升为 IL-6 或0.25 µg/毫升 IL-8。添加100µL 的稀释抗体每井。在室温下孵化水井2小时。</li>
		<li>通过吸入去除抗体溶液。用300µL 1x 洗涤缓冲器清洗每井4x。稀释链亲和素中的辣根过氧化物酶 (HRP), 浓度为0.05 µg/毫升。每井添加100µL 的链亲和素溶液。在室温下孵化水井30分钟。</li>
		<li>通过吸入去除抗体溶液。用300µL 1x 洗涤缓冲器清洗每井4x。添加100µL 的 3,3′, 5,5′-tetramethylbenzidine (TMB) 基质溶液, 每个井。在室温下孵化出20分钟的水井, 以允许颜色的发展。通过添加100µL 1 米的 HCl 停止溶液来停止反应。</li>
		<li>用 450 nm 的 ELISA 阅读器读出每个井的吸光度。</li>
		<li>为生成的标准绘制标准曲线。根据标准曲线计算条件介质中 IL-6 和 IL-8 的浓度。在对单元格计数进行规范化后绘制结果。计算重复样本的平均值。 注: ELISAs 应独立地重复至少 3x, 并且应根据这些结果计算平均值 (图 4)。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Hayflick, L., Moorhead, P. 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作者没有什么可透露的。

在这里, 我们提出了监测 WI-38 正常人成纤维细胞在 H-Ras 诱导衰老过程中胞内活性氧水平的方法。用细胞渗透性试剂 DCF-DA 和流式细胞仪定量测定活细胞内的活性氧水平。细胞吸收后, 脱乙酰度细胞内酯, 然后由 ROS 氧化, 形成高度荧光 2 ', 7 '-dichlorofluorescein (dcf)。用 FL1 检测器 (绿色荧光) 检测流式细胞仪可以探测到 DCF 荧光。利用 DCF-DA 染色法, 成功检测了 WI-38 正常人成纤维细胞在 H-Ras 诱导的细胞衰?...

50年前, 佛烈克和穆尔黑德发现, 正常细胞在细胞分裂1后, 在其增殖潜能耗尽后进入不可逆转的生长。这种现象现在被称为复制衰老, 并被认为与生物体衰老有强烈的关联2。虽然端粒的渐进侵蚀被认为是复制衰老的主要原因, 但据报道, 各种细胞应力, 如 DNA 损伤、致癌活化和氧化应激等, 都被报告诱发另一种细胞衰老。称为 "过早衰老" 或 "应力诱发衰老"。有趣的是, 过早衰老对肿瘤的激活起到了强有力的抑制作用, 如 H-Ras 和 BRAF。对小鼠模型和人体组织的研究已经产生了强有力的证据, 表明细胞衰老的生物标志物主要存在于前病变中, 在那里致癌 Ras 和 BRAF 被激活, 但在恶性癌症中被减少, 从这些病变3,4,5。除了它在衰老和肿瘤抑制方面的作用, 细胞衰老已经在以前的研究中显示, 在各种生理过程中发挥作用, 包括伤口愈合, 组织修复, 免疫监测, 胚胎发育6。
尽管生长骤停已被广泛研究为7细胞衰老的标志, 但大量的证据表明胞内活性氧 (ROS) 也有助于细胞衰老8。在不同类型的细胞衰老过程中, ROS 水平的升高, 包括复制衰老和癌基因引起的衰老, 最初是在数十年前的9,10。一个更直接, 外源治疗与致死剂量的 H2O2诱导衰老11,12。抑制 ROS 清除酶, 如 SOD1, 也导致过早衰老13。相比之下, 低环境氧条件和增加 ROS 清除延缓衰老的开始10,14,15。这些结果无疑表明, ROS 是细胞衰老诱导的重要调解人或决定因素。然而, ros 是如何促进细胞衰老的诱导以及在细胞衰老过程中 ros 水平的升高需要进一步的研究。
最近的研究表明, 衰老细胞通过 SASP16、17, 在邻近细胞和组织中具有强大的分泌活性。在衰老的组织中, 衰老细胞通过 SASP 的多种途径促进与年龄相关的组织功能障碍, 除了对增生细胞的自主耗尽之外。衰老细胞分泌的各种促炎因子, 如 IL-6、IL-8、TGFβ和基质金属蛋白酶 (金属蛋白酶), 通过组织稳态的损伤、组织结构的破坏, 引起与年龄相关的组织功能障碍,邻近细胞衰老, 无菌炎症18,19。然而, SASPs 可以产生有益的影响, 取决于生物环境。此外, SASPs 的 heterogenetic 性质取决于衰老细胞类型和细胞阶段, 强调需要进一步研究19。
在这里, 我们描述了快速和灵敏的细胞技术, 以评估细胞内活性氧的水平。此外, 还介绍了利用定量实时聚合酶链反应 (qPCR) 和 ELISA 法分析 SASP 因子的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>BOSC 23</td>
			<td>ATCC</td>
			<td>CRL-11269</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>wellgene</td>
			<td>LS202-02</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30013.02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>12800-082</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTI-MEM&#160;</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>pBabe puro-H-RasV12&#160;</td>
			<td>Addgene</td>
			<td>1768</td>
			<td></td>
		</tr>
		<tr>
			<td>pGAG/pol</td>
			<td>Addgene</td>
			<td>14887</td>
			<td></td>
		</tr>
		<tr>
			<td>pVSVG</td>
			<td>Addgene</td>
			<td>1733</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbofect</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0531</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td>8 mg/ml</td>
		</tr>
		<tr>
			<td>puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>2 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl &#946; D-galactopyranoside (X-gal)</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium ferrocyanide</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>wellgene</td>
			<td>LS015-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DCF-DA</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D6883</td>
			<td>10 mM&#160;</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>MMLV Reverse transcriptase</td>
			<td>Promega</td>
			<td>M1701</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green PCR master 2X mix</td>
			<td>Takara</td>
			<td>PR820A</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Primer</td>
			<td>Promega</td>
			<td>C118A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure distilled water</td>
			<td>Invitrogen</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6 ELISA assay</td>
			<td>PeproTech</td>
			<td>#900-TM16</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8 ELISA assay</td>
			<td>PeproTech</td>
			<td>#900_TM18</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>16555</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>BEMIS&#160;</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON</td>
			<td>TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Bioscience</td>
			<td>LSR Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4ml</td>
			<td>Merk Millipore</td>
			<td>UFC800324</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometer</td>
			<td>BioDrop</td>
			<td>80-3006-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR System</td>
			<td>Applied Biosystems</td>
			<td>ABI Prism 7500</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA Reader</td>
			<td>Molecular Devices</td>
			<td>EMax microplate reader</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative measurement of reactive oxygen species and senescence-associated secretory phenotype in normal human fibroblasts during oncogene-induced senescence

细胞衰老被认为是一种不可逆的生长状态, 在衰竭的增生能力或接触到各种压力。最近的研究扩大了细胞衰老在各种生理过程中的作用, 包括发育, 伤口愈合, 免疫监测和年龄相关的组织功能障碍。虽然细胞周期骤停是细胞衰老的一个重要标志, 但增加的细胞内活性氧 (ROS) 的生产也被证明对细胞衰老的诱导起着重要作用。此外, 最近的研究表明衰老细胞通过衰老相关的分泌表型 (SASP) 在邻近细胞和组织中表现出强有力的分泌活动。对细胞衰老的治疗策略的兴趣急剧增加强调需要对衰老机制, 包括胞内活性氧和 SASP 进行精确的了解。在这里, 我们描述了用 ros 敏感荧光染料和流式细胞仪定量评估 h-Ras 诱导的细胞衰老过程中细胞内 ROS 含量的协议。此外, 我们还介绍了敏感技术, 以分析诱导的 mRNA 表达和分泌的 SASP 因素。这些协议可应用于各种细胞衰老模型。

图 1显示了 H-Ras 引起的衰老的例子。WI-38 正常人成纤维细胞感染 H-RasV12 逆转录病毒诱发戏剧性形态学改变 (图 1B)。此外, 如图 1C所示, 在 H-RasV12 表达时, SA β-gal 染色活性显著增加。在 H-RasV12 逆转录病毒感染后, 超过70% 的细胞显示 SA β-gal 染色活性 6 d, 表明 H-RasV12 的表达成功地诱导 WI-38 细胞的细胞衰老, 正...

Watch this Scientific Journal Video about A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senesc

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

胞内活性氧在细胞衰老诱导中起着重要作用。在这里, 我们描述了一个敏感的测定细胞衰老过程中的 ROS 水平。我们还提供了评估衰老相关分泌表型的协议, 据报告, 这有助于各种年龄相关的功能障碍。

基因型衰老过程中正常人成纤维细胞活性氧和衰老相关分泌表型的定量测定

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

a-quantitative-measurement-reactive-oxygen-species-senescence

Research

JoVE Journal

Developmental Biology

8.0K 次观看.  Dong-A University. 胞内活性氧在细胞衰老诱导中起着重要作用。在这里, 我们描述了一个敏感的测定细胞衰老过程中的 ROS 水平。我们还提供了评估衰老相关分泌表型的协议, 据报告, 这有助于各种年龄相关的功能障碍。

视频: A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

从非转化人成纤维细胞增生四倍体细胞的建立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa is of high importance in any species, but these methods are rarely used and even less in teleost. Cryopreservation is a useful technique in aquaculture for different purposes, including gene banking and guaranteed sperm availability throughout the year. Freezing/thawing procedures could cause ROS production and damage the sperm cells. Considering the prospective damage that an excess of ROS production could cause in spermatozoa depending on their localization, here a detailed methodology to detect H2O2 and to evaluate its intracellular localization by confocal microscopy is provided. For this purpose, a combination of 3 fluorochromes (2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA), a live mitochondria stain and 4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)) are used to evaluate the co-localization of H2O2 with spermatozoa nuclei or mitochondria in Solea senegalesis sperm samples.

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

This protocol describes a detailed methodology for detecting H2O2 localization within Solea senegalensis spermatozoa using a sensitive fluorochrome DCFH-DA for ROS, a live mitochondria stain for mitochondria, and DAPI for nuclei visualization, respectively. The protocol is designed to be performed within 2 h with either fresh or thawed spermatozoa.

Solea Senegalensis精子中活性氧的细胞内定位评价

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径<em&gt;爪蟾</em&gt;胚胎发育

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

表征在拟南芥胚方法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

人肠道 Organoids 上皮屏障通透性的实时测量

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...