我们感谢 Demaria 实验室的成员进行了卓有成效的讨论, 并 Thijmen van 弗利特共享关于紫外线诱发衰老的数据和协议。

1. 一般准备
<ol><li>准备 D10 培养基。补充 DMEM 中 Glutamax 与10% 血清和1% 青霉素/链霉素 (最后浓度: 100 U/毫升)。</li>
	<li>准备无菌 PBS。根据制造商的指示, 在水中溶解药片。用高压釜消毒。</li>
	<li>准备1x 胰蛋白酶。稀释5毫升的胰蛋白酶-Versene EDTA/10x 1:10 在45毫升的无菌 PBS。 注意: 在整个协议中, 我们使用的细胞培养条件, 更接近的生理条件, 主要成纤维细胞。这意味着, 我们孵化的细胞在37摄氏度和 5% CO2通常做, 但使用5% O2 而不是 "标准" 20% O2.
</li>
	<li>使用层流罩、实验室外套和手套处理所有灭菌条件下的样品。仅在处理时将单元格保持在 20% O2 (房间条件)。</li>
</ol>2. 衰老的诱导
<ol><li>复制衰老
	<ol>
<li>在处理细胞或任何与它们接触的材料 (滴管、烧瓶、介质等) 的情况下, 使用层流罩、实验室外套和手套在无菌条件下工作。仅在处理时将单元格保持在 20% O2 (房间条件)。</li>
		<li>种子 7 x 105可行的主要成纤维细胞在低人口加倍 (PD) 在一个 T75 烧瓶 (~ 9.3 x 103细胞/cm2) 包含10毫升的 D10。</li>
		<li>在细胞孵化器中生长37摄氏度的细胞, 5% CO2和 5% O2直到它们达到70–80% 汇合 (在低 PD 中增殖培养的3–4天)。</li>
		<li>使用3毫升1x 胰蛋白酶和孵化的细胞培养孵化器在37°c 与 5% CO2和 5% O25–7分钟。用细胞培养显微镜定期监测细胞, 检查分离过程。</li>
		<li>当细胞为球形时, 通过加入9毫升的 D10 来阻止胰蛋白酶的反应。不要孵化超过10分钟。</li>
		<li>旋转细胞在 300 x g 5 分钟的细胞将形成一个颗粒在底部的管, 而碎片和较小的颗粒将留在上清。</li>
		<li>移除上清液并将细胞颗粒溶解在1毫升的 D10 中, 并根据制造商的指示或 Neubauer 室进行手动计数, 并使用自动单元计数器进行细胞计数。在计数时, 包括检测细胞的生存能力 (例如, 台盼蓝排除23)。</li>
		<li>使用此公式计算累计 PD: PD新= 3.32 * (日志 (细胞数总计)-(日志 (细胞数种子) + PDold 细胞总数 = 所有细胞计数: 死和活。 细胞数种子 = 可存活细胞的数量 (8x105细胞)。 PD老= 在播种的那一刻人口加倍。 PD新= 人口加倍在计数的时刻 (在孵化以后)。 注: 如果 7 x 105原发性成纤维细胞 (PD 35.2) 在 T-75 瓶上播种, 并且, 在4天他们到达80% 汇合并且再被分裂, 现在计数 1.3 x 106总细胞 (死 + 活)。 PD新= 3.32 * (日志 (130万个单元格)-日志 (70万)) + 35。2 PD新= 36。1</li>
		<li>补种 8 x 105细胞在一个新的 T75, 重复步骤 2.1. 2–2.1. 8。 注意: 经过多次连续的通道, 在细胞停止分裂之前, 这种文化需要更长的时间才能汇合。一旦细胞停止分裂, 测试衰老标记和/或收获的下游应用。</li>
		<li>考虑到衰老细胞的大小可能会使这种文化显得丰满, 但细胞数量却很低。因此, 当 PD 稳定和其他衰老标记出现在文化中时, 可以假设衰老 (参见协议 3.1–3.5)。 注: 使用增殖的主成纤维细胞作为控制。</li>
	</ol></li>
	<li>电离辐射引起的衰老
	<ol>
<li>在处理细胞或任何与它们接触的材料 (滴管、烧瓶、介质等) 的同时, 使用层流罩、实验室外套和手套在无菌条件下工作。仅在处理时将单元格保持在 20% O2 (房间条件)。</li>
		<li>种子 7 x 105可行的主要成纤维细胞在一个低 PD 在一个 T75 瓶 (~ 9.3 x 103细胞/cm2) 包含10毫升的 D10。</li>
		<li>一夜之间孵化细胞培养孵化器在37°c 与 5% CO2和 5% O2。</li>
		<li>根据使用中机器的说明, 将细胞暴露在伽玛照射下10 灰。</li>
		<li>从细胞中吸取培养基, 并用10毫升的 D10。</li>
		<li>在10毫升的细胞培养孵化器中孵育细胞在37°c 与 5% CO2和 5% O2为另外10天通常, 大约每3天替换媒介 D10。</li>
		<li>10天后, 测试衰老标记和/或使用细胞的下游应用。 注: 使用同一 PD (辐照前) 增殖的原发成纤维细胞作为控制。</li>
	</ol></li>
	<li>紫外线 (紫外线) 辐射引起的衰老
	<ol>
<li>在处理细胞或任何与它们接触的材料 (滴管、烧瓶、介质等) 的同时, 使用层流罩、实验室外套和手套在无菌条件下工作。仅在处理时将单元格保持在 20% O2 (房间条件)。</li>
		<li>种子 1.5–2 x 105可行的初级成纤维细胞在一个低 PD 在一个6井板 (1.5–2.0 x 104细胞/cm2), 增加2毫升的 D10 培养基。</li>
		<li>放置细胞培养孵化器在37°c 与 5% CO2和 5% O2和允许他们坚持到塑料至少 5 h。</li>
		<li>从细胞中脱下培养基。把6井板放在紫外线辐射室中间, 然后把塑料盖子摘下来。照射与 UVB, 20–30 mJ/cm2。</li>
		<li>每井加2毫升培养基。</li>
		<li>在2毫升的细胞培养孵化器中孵育细胞在37°c 与 5% CO2和 5% O2为另外7天通常, 大约每3天替换媒介 D10。 注: 7 天后, 细胞可以测试衰老标志物, 用于下游应用。使用相同 PD (辐照前) 的增殖原发成纤维细胞作为控制。</li>
	</ol></li>
	<li>阿霉素致衰老
	<ol>
<li>准备1,000x 阿霉素库存解决方案: 在 1x PBS 制造250µM 的阿霉素, 过滤-消毒溶液和整除500µL 每不育管。将阿霉素储存在-80 摄氏度。</li>
		<li>在处理细胞或任何与它们接触的材料 (滴管、烧瓶、介质等) 的同时, 使用层流罩、实验室外套和手套在无菌条件下工作。仅在处理时将单元格保持在 20% O2 (房间条件)。</li>
		<li>种子 7 x 105可行的主要成纤维细胞在一个低 PD 在一个 T75 瓶 (~ 9.3 x 103细胞/cm2) 包含10毫升的 D10。</li>
		<li>一夜之间孵化细胞培养孵化器在37°c 与 5% CO2和 5% O2。</li>
		<li>稀释11µL 的1,000x 阿霉素股票溶液在11毫升的 D10 到最后浓度250 nM.
</li>
		<li>从细胞中吸取培养基, 用10毫升的 D10 + 阿霉素代替。</li>
		<li>培养细胞为确切地 24 h 在细胞文化孵化器在37°c 与 5% CO2和 5% O2。</li>
		<li>从细胞中吸取培养基, 用10毫升的 D10 仔细冲洗一次。</li>
		<li>在10毫升的 D10 中孵化细胞6天, 定期更换培养基, 大约每3天进行一次。</li>
		<li>在7天, 测试衰老标记和/或用于下游应用。 注: 作为控制, 使用相同 PD 的增殖原发成纤维细胞治疗24小时与车辆 (PBS) 1:1, 000 在 D10。</li>
	</ol></li>
	<li>氧化应激引起的衰老
	<ol>
<li>在处理细胞或任何与它们接触的材料 (滴管、烧瓶、介质等) 时, 使用层流罩、实验室外套和手套在无菌条件下工作。仅在处理时将单元格保持在 20% O2 (房间条件)。</li>
		<li>种子 7 x 105可行的主要成纤维细胞在一个低 PD 在一个 T75 瓶 (~ 9.3 x 103细胞/cm2) 包含10毫升的 D10。</li>
		<li>一夜之间孵化细胞孵化器在37°c 与 5% CO2和 5% O2。</li>
		<li>在11毫升的 D10 中加入 22.6 ul 30% 过氧化氢, 在 D10 介质中制备200微米的过氧化氢溶液。 注: 通过作出曲线剂量-反应来评估毒性, 优化细胞类型的治疗。</li>
		<li>从细胞中吸取培养基, 加入10毫升的新制备的 D10 培养基 + 过氧化氢。孵育2小时在37°c 与 5% CO2和 5% O2。</li>
		<li>从细胞中吸取培养基, 用新鲜的 D10 清洗一次,不含过氧化氢。</li>
		<li>添加10毫升的 D10没有过氧化氢。</li>
		<li>孵育48小时在细胞培养孵化器在37°c 与 5% CO2和 5% O2。</li>
		<li>重复步骤 2.5. 4–2.5 8 次, 共三项治疗。</li>
		<li>检查下游应用的衰老标志或收获。 注: 作为对照, 使用 D10 中22.6 µL 的无菌水在2小时内治疗同一 PD 的增殖细胞。</li>
	</ol></li>
	<li>Epigenetically 引起的衰老
	<ol>
<li>根据表 1准备用于小分子的库存和工作解决方案。过滤-消毒解决方案。整除在无菌管中。存储在-20 摄氏度。</li>
		<li>在处理细胞或任何与它们接触的材料 (滴管、烧瓶、介质等) 的情况下, 在无菌条件下工作, 例如使用层流罩、实验室外套和手套。仅在处理时将单元格保持在 20% O2 (房间条件)。</li>
		<li>种子 7 x 105可行的主要成纤维细胞在一个低 PD 在一个 T75 瓶 (~ 9.3 x 103细胞/cm2) 包含10毫升的 D10。</li>
		<li>一夜之间孵化细胞培养孵化器在37°c 与 5% CO2和 5% O2。</li>
		<li>准备11毫升的 D10 中等 + 工作解决方案, 为理想的治疗。每治疗的确切稀释可以在表 1中看到。<ol>
<li>在11毫升的 D10 中准备11µL 1 毫米的萨哈工作溶液。</li>
			<li>在11毫升的 D10 中制备44µL 1 米丁酸钠工作溶液。</li>
			<li>在11毫升的 D10 中准备11µL 10 毫米 5 aza 工作溶液。 注意: 例如, 对细胞类型的兴趣进行优化处理, 比如对毒性进行曲线剂量反应。</li>
		</ol>
</li>
		<li>添加 D10 中 + 工作解决方案, 为所需的文化治疗。</li>
		<li>孵育24小时在细胞培养孵化器在37°c 与 5% CO2和 5% O2。</li>
		<li>重复步骤 2.6. 4–2.6 6 倍, 总共三种治疗方法。</li>
		<li>改变培养基为简单的 D10 没有药物。</li>
		<li>3天后, 细胞变得衰老, 并准备测试衰老标志物和下游应用。 注: 作为对照, 使用 D10 中 + 车治疗三天的同一 PD 的增殖性原发成纤维细胞。在这三天内, D10 中 + 车需要每24小时刷新一次。该车取决于所使用的治疗方法: 1:1, 000 亚砜为萨哈和 5-aza 和1:250 无菌水的丁酸钠。</li>
	</ol></li>
</ol>3. 衰老的标志物
<ol><li>制备
	<ol>
<li>准备20毫克/毫升 x-加仑: 溶解20毫克的 x 加仑在1毫升的甲基酰胺或亚砜。贮存在-20 摄氏度, 免受光照。</li>
		<li>准备 0.1 M 柠檬酸溶液: 在100毫升的水中溶解2.1 克柠檬酸一水合物。在室温下贮存。</li>
		<li>制备0.2 米磷酸钠溶液: 溶解2.84 克磷酸二钠或3.56 克磷酸二钠, 在100毫升水中脱水。在室温下贮存。</li>
		<li>准备0.2 米柠檬酸/磷酸磷酸钠 pH 值 6.0: 溶解36.85 毫升 0.1 M 柠檬酸溶液和63.15 毫升的0.2 米磷酸钠。完全调整到 pH 值 6.0。在室温下贮存。</li>
		<li>准备100毫米盐钾: 盐50毫升水中溶解2.1 克钾。贮存在4摄氏度, 免受光照。</li>
		<li>准备100毫米氰化钾钾: 氰化钾50毫升水中溶解1.7 克钾。贮存在4摄氏度, 免受光照。</li>
		<li>准备5米氯化钠: 在50毫升水中溶解14.6 克氯化钠。在室温下贮存。</li>
		<li>准备1米氯化镁: 在50毫升的水中溶解4.8 克氯化镁。在室温下贮存。</li>
		<li>在 pbs 中制备2% 甲醛 + 0.2% 戊二醛: 在 pbs 的 12.5 ul 中溶解800µL 25% 戊二醛和16% µL 100 甲醛。在室温下贮存, 免受光线的侵害。</li>
		<li>根据 表 2准备新的染色液。
</li>
	</ol></li>
	<li>衰老相关β-乳糖酶染色
	<ol>
<li>对于每个样本, 种子 1 x 104细胞在24井板 (5.2 x 103细胞/cm2) 的至少一个井中包含500µL 的 D10, 使细胞稀疏。治疗 (如果适用的话) 可以直接在这个盘子上进行, 或者, 已经被治疗的细胞可以被重新播种到24井板中。</li>
		<li>一夜之间孵化细胞在37°c 与 5% CO2和 5% O2。</li>
		<li>用 500 ul 的 PBS 冲洗细胞两次。</li>
		<li>在室温下使用 500 ul/井2% 甲醛 + 0.2% 戊二醛在 PBS 中固定3–5分钟。</li>
		<li>用 500 ul 的 PBS 冲洗细胞两次。</li>
		<li>准备新的染色液, 根据样品的数量进行染色。</li>
		<li>添加染色液 (500 µL/井), 用 parafilm 密封板, 避免蒸发。 注: 蒸发可能导致晶体形成和阻碍观察在显微镜下。</li>
		<li>在黑暗中孵化细胞 (例如用铝箔覆盖) 在干燥的孵化器 (没有 CO2) 为 12–16 h 的37摄氏度。 注: CO2可能影响 pH 值, 因此修改结果。某些细胞类型可能需要更短的孵化时间。</li>
		<li>洗两次与 500 ul 的 PBS。</li>
		<li>评估结果。阳性细胞在正常的光显微镜下呈现蓝色 (主要是) 核周染色 (图 1A)。</li>
		<li>为定量, 观察每个样本至少100个细胞和计数阳性细胞的数量。由于衰老细胞改变形状, 通常很难定义细胞边界和计数细胞。Counterstain 与 DAPI, 以促进个别细胞的可视化和量化 (图 1B)。使用荧光显微镜 (图 1C) 量化样本 (阳性细胞与总数量的百分比)。拍摄同一样本的多个图片, 以便在最后你可以数至少100单细胞和评估 SA β-gal 阳性细胞的百分比。</li>
		<li>比较衰老细胞的结果与相应的治疗方法的对照。 注: 在同一个样本上与教育局的共同染色是可能的。认为细胞应该在盖玻片上播种。</li>
	</ol></li>
	<li>留学合并化验
	<ol>
<li>根据样品数量, 将盖玻片/井放在24井板上进行评估。</li>
		<li>种子 1 x 104细胞在一个24井板 (5.2 x 103细胞/cm2) 的至少一个井中, 每个条件包含500µL 的 D10, 使细胞稀疏。治疗 (如果适用的话) 可以直接在这个盘子上进行, 或者, 已经处理过的细胞可以被重新播种到24井板中。</li>
		<li>一夜之间孵化细胞在37°c 与 5% CO2和 5% O2。</li>
		<li>根据要处理的样品数量 (每样品250个 ul), 在 D10 (1:250) 中制作20µM 解决方案。</li>
		<li>从每个井中取出一半的介质 (250 ul), 用刚刚准备好的 D10 解决方案来处理并替换它。最终的µM 浓度是10。</li>
		<li>孵化 18–24 h 在细胞培养孵化器在37°c 与 5% CO2和 5% O2。在控制和衰老细胞中使用相同的孵化时间。</li>
		<li>用 500 ul 的 PBS 洗涤2x。</li>
		<li>固定细胞为10分钟与 500 ul 4% 甲醛在 PBS。</li>
		<li>孵育5分钟在 500 ul 100 毫米 (pH 7.6)。</li>
		<li>Permeabilize 10 分钟的细胞在 500 ul 0.1% 海卫 X-100 在 PBS。</li>
		<li>用 PBS 洗3x。</li>
		<li>为每个盖玻片准备 50 ul 标签组合, 按以下顺序添加组件: 44.45 µL 的 PBS, 0.5 µL 铜 (II),4, 0.05 µL sulfo-Cy3-azide, 5 µL 钠抗坏血酸。</li>
		<li>把 50 ul 的标签组合在一块 parafilm。用一双镊子和一根针举起盖玻片, 让它在标签混合的顶部休息, 表面上含有向下的细胞。确保没有气泡, 盖玻片的整个表面都在接触标签组合。在黑暗中孵化30分钟。</li>
		<li>把这些细胞放回24井板井中, 用 PBS 冲洗三次。</li>
		<li>安装与安装介质 (包括 DAPI, 以可视化原子核) 到玻璃幻灯片, 让他们干一夜。</li>
		<li>使用荧光显微镜可视化了该公司的注册。使用适合 Cy3 的过滤器 (激发/发射: 552/570 nm)。</li>
		<li>拍摄细胞的多张图片, 以便在每个条件下至少有100个细胞 (Cy3 和 DAPI) 进行量化。</li>
		<li>使用以下公式量化纳入教育局的单元格百分比: 教育局阳性细胞 (%) = (教育局阳性细胞计数 (Cy3)/总细胞计数 (DAPI)) * 100</li>
		<li>比较衰老细胞的结果与相应的治疗方法的对照。 注: 在同一样品上与 SA β-gal 进行共染色是可能的。认为细胞仍应在盖玻片上播种。</li>
	</ol></li>
	<li>p16、p21 和 SASP 的基因表达
	<ol>
<li>准备一组感兴趣的基因 (50 µM 每个底漆)。 注: p16、p21 及相关 SASP 因素的 qPCR 是衰老状态的信息。本文所描述的协议使用通用探针库 (价目表) 系统进行相对量化, 使用实时 PCR。表 3显示了用于检测 CDK 抑制剂 p16 和 p21 以及最相关 SASP 成员的底漆的概述, 以及蛋白和肌动蛋白, 作为该化验的参考基因。表 3的最后一列登记用于每种化验的特定价目表探针。</li>
		<li>为所需的化验准备单独的 qPCR 反应组合。总是包括参考基因以及根据表 4。</li>
		<li>以重复或三份运行所有示例。</li>
		<li>负载7.5 µL/井的 qPCR 反应混合在一个384井板上。</li>
		<li>添加〜5的 cDNA 溶解在2.5 µL 的无 RNase 水。 注意: 最好有类似数量的 cDNA 的所有样本进行比较。这可以通过使用相同数量的 RNA 来实现反向转录。</li>
		<li>用密封盖住盘子, 确保它正确地覆盖在盘子上的所有油井。</li>
		<li>旋转板在 2000 x g 1 分钟。</li>
		<li>使用以下协议在 Lightcycler 中运行40个周期的板块: 95°c 为7分钟 40周期95°c 为 5 s 和60°c 为三十年代 37°c 为1分钟</li>
		<li>为了对结果进行分析, 使用建议的方法对 Livak 和同事分析 qPCR 数据24。使用蛋白或肌动蛋白作为参考基因计算ΔCt 值, 并使用适当的控制来计算ΔΔCt 价值的特定的衰老诱导治疗。</li>
	</ol></li>
	<li>IL6 蛋白的表达与分泌
	<ol>
<li>种子 5–10 x 104细胞在6井板 (5.2–10.5 x 103细胞/cm2) 的至少一个井中, 每个条件包含2毫升的 D10。治疗 (如果适用的话) 可以直接在这个盘子上进行, 或者, 已经被治疗的细胞可以被重新播种到24井板中。让它在播种后一夜之间站立。</li>
		<li>取出培养基并更换2毫升的 DMEM 培养基,不加血清。正常条件下孵育24小时。</li>
		<li>在15毫升管中收集培养基。</li>
		<li>离心机样品在 300 x g 为5分钟培养基可以保存在-80 °c, 直到处理。</li>
		<li>按照制造商的指示执行 ELISA。</li>
		<li>衰老细胞相对于特定衰老诱导治疗的适当控制结果比较</li>
	</ol></li>
</ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forging+a+signature+of+in+vivo+senescence.">Forging a signature of in vivo senescence.</a> Nature Reviews Cancer. 15 (7), 397-408 (2015).</li><li>Correia-Melo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+are+required+for+pro-ageing+features+of+the+senescent+phenotype.">Mitochondria are required for pro-ageing features of the senescent phenotype.</a> The EMBO Journal. 10, e201592862 (2016).</li><li>Loaiza, N., Demaria, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+and+tumor+promotion:+Is+aging+the+key?.">Cellular senescence and tumor promotion: Is aging the key?.</a> Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. , (2016).</li><li>Coppe, J. 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这里解释的协议被优化了为人的主要成纤维细胞, 特别是 BJ 和 WI-38 细胞。复制衰老, 电离辐射和阿霉素的协议已成功地应用于其他类型的成纤维细胞 (HCA2 和 IMR90) 和其他细胞类型 (即新生儿黑色素细胞和角质形成素或 iPSC 衍生心肌细胞)在我们的实验室里。然而, 可以通过调整一些细节, 如种子细胞数量, 方法和化学物质来帮助细胞附着/分离到塑料支架上, 以及治疗的剂量以避免毒性, 来优化其他细?...

在 1961年, 佛烈克和穆尔黑德报告说, 在连续1段后, 培养的主要成纤维细胞失去了增殖潜能。这一过程是由每细胞分裂后端粒的顺序缩短引起的。当端粒到达临界短的长度时, 它们被 DNA 损伤反应 (DDR) 所识别, 它激活了不可逆转的增殖--也被定义为复制衰老。复制衰老是目前已知的许多刺激之一, 导致一个永久性细胞周期逮捕的状态, 使细胞不敏感的 mitogens 和凋亡信号2,3。衰老程序通常的特点是额外的功能, 包括高溶酶活性, 线粒体功能障碍, 核变化, 染色质重组, 内质网应力, DNA 损伤和衰老相关分泌表型 (SASP)3,4。衰老细胞在体内具有多种功能: 发育、创面愈合和肿瘤抑制2。同样, 众所周知, 它们在衰老和肿瘤进展5中起着重要的作用。衰老的消极和部分矛盾的影响通常归因于 SASP6。
最近, 它表明, 消除衰老细胞的小鼠导致寿命延长和消除许多老化功能7,8,9,10,11,12. 同样, 已经开发了多种药物, 以消除衰老细胞 (senolytics) 或靶向 SASP13,14。抗衰老治疗潜力最近引起了人们的广泛关注。
对细胞衰老相关机制的研究和药理干预的筛选严重依赖于体外模型, 特别是在人原代成纤维细胞中。虽然不同的衰老诱导因子激活了一些共同的特征, 但衰老表型的大变异性被观察到, 并依赖于各种因素, 包括细胞类型, 刺激和时间点3,15, 16,17。研究和定位衰老细胞的异质性是当务之急。因此, 本议定书的目的是提供一系列方法, 以诱发衰老的主要成纤维细胞使用不同的治疗。正如它将解释, 这些方法可以很容易地适应其他细胞类型。
除了复制衰老, 我们还描述了其他五种衰老诱导治疗: 电离辐射, 紫外线 (紫外线) 辐射, 阿霉素, 氧化应激和后生变化 (即促进组蛋白乙酰化或 DNA 去甲基化).电离辐射和紫外线辐射直接导致 DNA 损伤, 并在适当剂量下引发衰老18,19。阿霉素还导致衰老主要是通过锂 dna 损伤的 dna 和干扰拓扑异构酶 II 功能, 从而停止 DNA 修复机制20。衰老的关键基因表达通常由组蛋白乙酰化和 DNA 甲基化控制。因此, 组蛋白乙酰抑制剂 (如丁酸钠和萨哈) 和 DNA 去甲基化 (例如, 5-aza) 剂会导致正常细胞21,22的衰老。
最后, 与衰老细胞相关的四种最常见的标记将被解释为: 衰老相关的乳糖酶 (SA β) 的活性、由免疫组织测定的 DNA 合成率、细胞周期调节器的过度表达和细胞周期蛋白依赖性激酶抑制剂 p16 和 p21, 并表达和分泌的 SASP 成员。

<table>
	<tbody>
		<tr>
			<td>DMEM Media - GlutaMAX</td>
			<td>Gibco</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S; 10,000 U/ml)</td>
			<td>Lonza</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>SC-202581</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water (not DEPC-Treated)</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Sarstedt</td>
			<td>833911002</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA Solution</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Gibco</td>
			<td>18912-014</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>T9661-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;15 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430791</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Sarstedt</td>
			<td>83.3920</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Sarstedt</td>
			<td>83.3922</td>
			<td></td>
		</tr>
		<tr>
			<td>13mm round coverslips</td>
			<td>Sarstedt</td>
			<td>83.1840.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip</td>
			<td>Merck Chemicals</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Cesium137-source</td>
			<td></td>
			<td></td>
			<td>IBL 637 Cesium-137&#947;-ray machine</td>
		</tr>
		<tr>
			<td>UV radiation chamber</td>
			<td></td>
			<td></td>
			<td>Opsytec, Dr. G&#246;bel BS-02</td>
		</tr>
		<tr>
			<td>Doxorubicin dihydrochloride&#160;</td>
			<td>BioAustralis Fine Chemicals</td>
			<td>BIA-D1202-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>7722-84-1</td>
			<td></td>
		</tr>
		<tr>
			<td>5-aza-2&#8217;-deoxycytidine</td>
			<td>Sigma-Aldrich</td>
			<td>A3656</td>
			<td></td>
		</tr>
		<tr>
			<td>SAHA</td>
			<td>Sigma-Aldrich</td>
			<td>SML0061</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Butyrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal (5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside)</td>
			<td>Fisher Scientific</td>
			<td>7240-90-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>5949-29-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dibasic phosphate</td>
			<td>Acros organics</td>
			<td>7782-85-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide&#160;</td>
			<td>Fisher Scientific</td>
			<td>14459-95-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferricyanide</td>
			<td>Fisher Scientific</td>
			<td>13746-66-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Merck Millipore</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Fisher Chemicals</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>25% glutaraldehyde</td>
			<td>Fisher Scientific</td>
			<td>111-30-8,7732-18-5</td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde (w/v)</td>
			<td>Thermo-Fisher Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU (5-ethynyl-2&#8217;-deoxyuridine)</td>
			<td>Lumiprobe</td>
			<td>10540</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-Cyanine3 azide (Sulfo-Cy3-Azide)</td>
			<td>Lumiprobe</td>
			<td>D1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) sulfate pentahydrate (Cu(II)SO4.5H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>209198</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Acros organics</td>
			<td>215682500</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS base</td>
			<td>Roche</td>
			<td>11814273001</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 Multiwell Plate 384, white&#160;</td>
			<td>Roche</td>
			<td>4729749001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightcycler 480 sealing foil&#160;</td>
			<td>Roche</td>
			<td>4729757001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sensifast Probe Lo-ROX kit&#160;</td>
			<td>Bioline</td>
			<td>BIO-84020</td>
			<td></td>
		</tr>
		<tr>
			<td>UPL Probe Library</td>
			<td>Sigma-Aldrich</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6 DuoSet ELISA</td>
			<td>R&#38;D</td>
			<td>D6050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad TC20</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides</td>
			<td>Bio-Rad</td>
			<td>145-0017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry incubator</td>
			<td>Thermo-Fisher Scientific</td>
			<td>Heratherm</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>Merck Millipore</td>
			<td>1.10983</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm &#39;M&#39; laboratory film</td>
			<td>Bemis&#160;</td>
			<td>#PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and validation of cellular senescence in primary human cells

细胞衰老是一种永久性细胞周期停止激活的反应, 以应对不同的破坏性刺激。细胞衰老的活化是各种病理生理条件的标志, 包括肿瘤抑制、组织重塑和衰老。细胞衰老的诱导体在体内仍然缺乏特征。然而, 一些刺激可以用来促进细胞衰老前体。其中, 最常见的衰老诱导因子是复制衰竭, 电离和非电离辐射, 毒性药物, 氧化应激, 去甲基化和乙酰剂。在这里, 我们将提供有关如何使用这些刺激诱导成纤维细胞衰老的详细说明。该协议可以很容易地适应不同类型的主要细胞和细胞系, 包括癌细胞。我们还描述了不同的方法来验证衰老诱导。特别是, 我们专注于测量溶酶体与衰老相关的β-乳糖酶 (SA β-加仑) 的活性, 使用 5-乙炔-2 '-脱氧尿苷 () 的 DNA 合成率, 细胞周期的表达水平抑制剂 p16 和 p21, 以及衰老相关分泌表型 (SASP) 成员的表达和分泌。最后, 给出了实例结果, 并讨论了这些协议的进一步应用。

衰老成纤维细胞中 SA β-gal 染色的富集
β-乳糖酶 (β-加仑) 是一种酶, 表达在所有细胞和具有最佳 pH 值为 4.025,26。然而, 在衰老过程中, 溶酶体体积增大, 衰老细胞积累β-加仑。这种酶的增加使它有可能检测到它的活性, 即使在次优 pH 值 6.025,27?...

Watch this Scientific Journal Video about Induction and Validation of Cellular Senescence in Primary Human Cells at JoVE.com

Induction and Validation of Cellular Senescence in Primary Human Cells

原发性细胞衰老的诱导与鉴定

在这里, 我们讨论了一系列的诱导和确认细胞衰老在培养细胞的协议。我们专注于不同的衰老诱发刺激, 并描述了常见的衰老相关标记的量化。我们提供的技术细节, 以成纤维细胞作为模型, 但协议可以适应各种细胞模型。

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

induction-and-validation-of-cellular-senescence-in-primary-human-cells

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Developmental Biology

Unit 1

Cell Proliferation

SCIENTISTS IN ACTION

16.6K 次观看.  University of Groningen, University Medical Center Groningen. 在这里, 我们讨论了一系列的诱导和确认细胞衰老在培养细胞的协议。我们专注于不同的衰老诱发刺激, 并描述了常见的衰老相关标记的量化。我们提供的技术细节, 以成纤维细胞作为模型, 但协议可以适应各种细胞模型。

视频: Induction and Validation of Cellular Senescence in Primary Human Cells

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

诱导蛋白缺失的通过<em&gt;在子宫内</em&gt;电穿孔来定义赤字的神经细胞迁移的转基因模型

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

科研

发育生物学

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 ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

在肉瘤细胞间质上皮转变的诱导

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

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 ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

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

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

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. ...