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><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</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&amp;nbsp;</td><td>GIBCO</td><td>31985-070</td><td></td></tr><tr><td>pBabe puro-H-RasV12&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;beta; 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&amp;nbsp;</td><td>D6883</td><td>10 mM&amp;nbsp;</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>&lt;strong&gt;EQUIPMENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.45 &amp;mu;m syringe filter</td><td>sartorius</td><td>16555</td><td></td></tr><tr><td>Parafilm</td><td>BEMIS&amp;nbsp;</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

Universidad de Cartagena UDEC

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

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

量化衰老癌症细胞的敏感方法

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

科研

医学

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 ...

发育生物学

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

癌症研究

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

远红荧光衰老相关β-半乳糖苷酶探针，用于通过流式细胞术鉴定和富集衰老肿瘤细胞

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

生物化学

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

使用2'，7'-二氯荧光素双乙酸探针和流式细胞术在穆勒神经胶质细胞中定量活性氧

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

神经科学

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

生物学

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Detecting Reactive Oxygen Species

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are many deleterious effects associated with unchecked ROS production, including structural damage to DNA and other biological molecules. However, ROS can also be mediators of physiological signaling. There is accumulating evidence that ROS play significant roles in everything from activation of transcription factors to the mediation of inflammatory toxicity that kills foreign pathogens and defend the body.In this video we will delve into the associations between ROS, metabolism and disease. After establishing their significance, we will discuss the principles and a protocol of a commonly used methodology for measuring ROS levels in cells: the use of non-fluorescent probes that become fluorescent upon oxidation. Lastly, we will review some current applications of this technique in cell biology research.

Reactive Oxygen Species and Oxidative Stress

检测活性氧物种

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are ...