Name: flexible measurement of bioluminescent reporters using an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (alligator)
Uploaded: 2017-12-13T14:00:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

我们要感谢凯恩的研究工作, 与我们一起开发这个系统, 特别是马克-汉森, 格雷厄姆和 Correia。我们还感谢大卫威尔士和 Akhilesh 在设计阶段有价值的讨论, 以及彼得 Laskey (以前的滨) 安排贷款的演示相机和大卫王为他的关键投入的手稿。

1. 细胞的播种和温度夹带
注意: 该协议已被严格测试使用主要和永生的小鼠成纤维细胞表达 PERIOD2::LUCIFERASE (PER2::LUC) 融合蛋白4。可能需要对使用其他细胞系的实验进行调整。
<ol>
	<li>在开始细胞培养之前, 安置以下入37° c 水浴到温暖: 磷酸盐缓冲盐水 (pbs) (ph 7.4), pbs 补充与0.68 毫米乙酸酸, ph 7.2 (pbs + EDTA), 适当的细胞培养媒介,例如,Dulbecco 的改良鹰的培养基 (DMEM) 补充10% 胎牛血清 (FBS), 100 U/毫升青霉素和100µg/毫升链霉素, 0.25% 胰蛋白酶溶液。</li>
	<li>用 5ml PBS + EDTA 溶液稀释胰蛋白酶 1:3, 回到水浴。</li>
	<li>采取125厘米2烧瓶的荧光表达细胞, 是70-90% 汇合。吸媒体。添加10毫升 5ml PBS。</li>
	<li>吸入 PBS添加2.5 毫升5ml 胰蛋白酶-EDTA, 并将烧瓶返回到37° c 孵化器5分钟。</li>
	<li>将细胞从孵卵器中取出, 然后在显微镜下观察。一旦大多数细胞从瓶底分离出来, 继续下一步。如果大多数细胞仍然附着在烧瓶的底部, 把它归还给孵化器, 直到大多数细胞处于悬浮状态。</li>
	<li>在烧瓶中加入7.5 毫升的标准细胞培养基。(可选) 根据需要移除1毫升以进一步通过细胞。</li>
	<li>使用例和台盼蓝计算剩余细胞的密度。进一步稀释细胞, 适当地为那细胞线。 注: 在下面的实验中, PERIOD2::LUC 成纤维细胞在 6 x 103细胞/cm2和 1 x 104细胞/cm2中的 96-井板, 35 mm 菜肴, 或通道幻灯片种子。</li>
	<li>种子细胞到组织培养皿或将用于记录的盘子。 注意: 建议使用黑色双面、透明底板, 这样可以在录制开始前评估文化健康状况, 同时减少记录期间井间的干扰。在生物发光水平极低的地方, 白色的盘子/盘子应该被用来最大化光线的检测, 尽管请注意, 磷光会导致持续数小时的背景信号的增加。</li>
	<li>将盘子返回孵化箱, 让单分子膜达到100% 汇合 (大约7天)。 注: 一旦汇合, 抑制接触的细胞系可在实验前维持三周, 前提是定期刷新介质 (每5-7 天)。</li>
	<li>一旦汇合, 同步细胞节律使用应用的温度周期 (12 小时在32° c, 12 h 在37° c) 为至少 72 h 在实验之前在试验5,6 (使用预编程的循环孵化器, 由通过串行 RS-232 端口连接到孵化器的计算机。 注意: 同步细胞节律所需的温度周期数可能因细胞线而异, 因此可能需要对其他细胞系进行优化。急性药理刺激使用佛司可林或地塞米松也已被用于同步细胞节律7,8。</li>
</ol>
2. NS21 制剂
注: NS21 是一种无血清补充剂, 用于维持神经细胞和其他细胞培养物。这是一个类似的补充称为 B27 的细化, 这是商业可用的, 可以作为一个血清置换在昼夜生物发光记录9。任何一种补充都可以在记录介质中交替使用, 用于下面描述的实验协议。NS21 in-house 是相当可行和成本效益的, 如陈et al.10, 如下所述。
<ol>
	<li>平衡在表 1中描述的所有组件在开始前的室温下为1小时。</li>
	<li>在324毫升的基培养基中溶解50克牛白蛋白 (如, neurobasal) 在冰上。尽可能少搅拌混合物。</li>
	<li>添加所有其他组件, 在每个组件后进行最小搅拌, 但仍可确保彻底混合。目的在90分钟内解散所有组件。</li>
	<li>分最后的混合物, 并储存在-20 ° c, 直到需要。避免重复的冻融循环。 注: 这种混合物太粘稠, 无法在这个阶段进行过滤, 但在将其添加到细胞之前, 会被介质稀释后被过滤。</li>
</ol>
3. 记录介质准备
注意: 生物发光孵化器的主要优势是其他设备的记录纵向生物发光是, 由于能够 humidify 孵化器和变化的部分压力, O2和 CO2, 它是可能使用更广泛的媒体条件来记录生物荧光-包括更接近于不同细胞类型体内的生理生态位的条件。下面我们描述了改编自黑斯廷斯et al.的记录介质的配方9, 我们经常使用培养成纤维细胞和其他细胞类型。第一个是密封培养条件 (没有气体交换), 第二个是生理相关的条件, 应在湿润条件下使用, 5% CO2。许多其他的变体都是可能的和可取的, 这取决于确切的应用和细胞类型。
<ol>
	<li>拖把缓冲高糖培养基的制备 注: 库存解决方案 (1 升):D 记忆粉 (8.3 克/升), 0.35 毫克/毫升碳酸氢钠, 5 毫克/毫升葡萄糖, 0.02 M 拖把, 100 U/毫升青霉素, 100 µg/毫升链霉素。<ol>
		<li>将8.3 克 DMEM 粉末溶解在900毫升的超纯水中, 在1000毫升的测量缸中搅拌30分钟直到所有颗粒溶解。</li>
		<li>加入50毫升葡萄糖溶液 (100 克/升), 20 毫升的拖把 (1 米, pH 7.6), 10 毫升的100x 笔/链球菌溶液, 并挑起进一步10分钟。</li>
		<li>加入4.7 毫升7.5% 碳酸氢钠溶液, 再搅拌5分钟。</li>
		<li>将介质 pH 值调整到 7.6 (室温时) 或 7.4 (37 ° c) 与 HCl/氢氧化钠。</li>
		<li>添加超纯水以产生1000毫升的最终体积。</li>
		<li>通过过滤0.22 µm 过滤器和存储在4° c, 直到需要消毒。</li>
		<li>在实验之前, 补充一个适当的数量的股票解决方案与10% 血清, 2mM l-丙-l-谷氨酰胺, 2% NS21, 和1毫米素, 使一个工作的股票。通过这个股票通过0.22 µm 无菌过滤器。</li>
		<li>使用 osmometer 测量介质的渗透。<ol>
			<li>打开 osmometer</li>
			<li>校准 osmometer, 首先用超纯水。在测量容器的底部加50µL 水。确保液体中没有气泡。将容器夹在探头上。按 "0", 并小心地把 osmometer 臂降到最低点。等到阅读完成后, 绿色的 "结果" 灯亮起, 然后再举起手臂并取出容器。</li>
			<li>重复校准 osmometer 300 mOsmol/千克使用50µL 校准标准。在放下手臂前请按 "Cal"。</li>
			<li>通过在测量容器的底部添加50µL 的介质来测量样品渗透, 并在上面测量。在降低 osmometer 臂前按 "样本"。</li>
		</ol>
		</li>
		<li>使用5米氯化钠将渗透调整到 350 mOsmol。</li>
	</ol>
	</li>
	<li>小贩3-缓冲低血糖介质 (灌注介质) 制备 注: 库存解决方案 (1 升): DMEM 粉 (8.3 克/升), 3.7 毫克/毫升碳酸氢钠, 1 毫克/毫升葡萄糖, 100 U/毫升青霉素, 100 µg/毫升链霉素。<ol>
		<li>将8.3 克 DMEM 粉末溶解在900毫升的超纯水中, 在1000毫升的测量缸中搅拌30分钟直到所有颗粒溶解。</li>
		<li>加入50毫升葡萄糖溶液 (100 克/升), 10 毫升的100x 笔/链球菌的解决方案, 并挑起进一步10分钟。</li>
		<li>加入49.4 毫升7.5% 碳酸氢钠溶液, 再搅拌5分钟。</li>
		<li>调整介质 pH 值至 7.6 (室温) 或 7.4 (37 ° c) 与 HCl/氢氧化钠。</li>
		<li>加入超纯水, 最终体积为1000毫升。</li>
		<li>通过一个0.22 µm 无菌过滤器和存储在4° c, 直到使用的解决方案。</li>
		<li>在实验之前, 补充一个适当的数量的股票解决方案与2% 血清, 2 毫米 l-丙-l-谷氨酰胺, 2% NS21 和1毫米素, 以使一个工作的股票。通过这个股票通过0.22 µm 无菌过滤器。</li>
		<li>测量和调整渗透到 350 mOsmol 使用5米氯化钠, 至于拖把缓冲介质。</li>
	</ol>
	</li>
</ol>
注意: 素浓度应根据每个细胞类型和上下文进行经验判断。有关详细信息, 请参阅菲尼et al.11血清和 NS21 (或 B27 如果使用) 浓度可以根据应用变化。然而, 我们确实建议, 除非经实证检验, 否则, 血清和 NS21 (或替代无血清补充剂) 使用, 因为这些促进细胞存活和依恋。在不接触抑制的细胞系中, 可能有必要降低记录介质中的血清浓度, 以防止增殖的混杂效应, 这也是由血清促进的。
4. 记录
<ol>
	<li>在开始录音之前, 放置适当的工作介质存货 (从步骤3.1 或3.2 取决于实验) 在一个37° c 水浴到温暖。</li>
	<li>当媒体变暖时, 将相机聚焦在生物发光孵化器中。
	<ol>
		<li>拧开 water-impermeable, 加热中性密度过滤器并预留。</li>
		<li>设置软件以记录高采集率的视频。点击 "收购 |购置设置 "。将 "曝光时间" 设置为0.2 秒, 动态循环时间为0.3 秒钟。确保已关闭 EM 增益。</li>
		<li>关闭 "购置设置" 窗口, 然后单击 "获取视频" 图标。</li>
		<li>使用聚焦气缸, 旋转相机镜头, 直到货架上的项目在高度被记录清楚地在视频焦点。</li>
		<li>通过单击 "停止视频" 图标停止视频。</li>
		<li>将加热后的中性密度过滤器拧回到位;不这样做将导致损坏的 EM CCD 相机和/或雾的目标, 由于凝结。</li>
	</ol>
	</li>
	<li>将 O2、CO2和温度设置为所需的实验条件, 使用生物发光孵化器前面的控制面板。 注: 如果在这个阶段执行灌注细胞培养直接进入5节。</li>
	<li>从组织培养箱中取出细胞, 从培养基中抽出。用5ml 的工作库存更换介质。</li>
	<li>如果在实验过程中不将生物发光培养箱加湿, 那么就用不透气的密封密封盘子和盘子。</li></ol>如果生物发光孵化器是要加湿的, 那么它不是严格地需要密封菜, 虽然它是可取的密封菜, 如 96-井板, 使用透气密封, 作为一个小数量的蒸发可以否则甚至在湿润的恒温箱中也会发生。 注: 加湿是通过在孵化器的底部填充水托盘来实现的。
	<li>将细胞转移到生物发光培养箱, 关闭孵化箱门, 确保孵化箱盖到位, 形成光密封。</li>
	<li>选择适合于应用程序的记录参数。
	<ol>
		<li>点击 "收购 |购置设置 "。</li>
		<li>在 "相机设置" 面板中, 设置: "获取模式" 到 "动态";' 曝光时间 ' 到所需曝光时间, 以秒为单位 (见下文注);' 积累 ' 到 1;' 动能系列长度 ' 到需要的承购周期的数量;' 动能循环时间 ' 到期望的成像间隔 (确保这是大于曝光时间);' 变速 ' 到4.33 µsecs;' 增益 ' 到 1;和 ' EM 增益 ' 到期望的水平 (见下面的说明)</li>
		<li>在 "自动保存" 面板中, 将文件类型设置为. sif 或. tif。提供文件名和适当的保存位置。</li>
		<li>在 "后台打印" 面板中, 将文件类型设置为 tiff。提供文件名和适当的保存位置。关闭 "购置设置" 窗口。</li>
	</ol>
	</li>
	<li>单击 "采取信号" 按钮开始录制。 注意: 由于生物发光孵化器可用于大量不同的细胞和组织类型, 所有这些都将有不同程度的生物发光, 所需的曝光时间收集适当的生物发光信号将不同实验.也可以改变成像的电子倍增增益, 以增加所采集信号的大小。对于未知亮度的新应用, 我们建议首先采用一个相对较短的曝光时间 (大约10分钟) 的单一图像, 而不需要 em 增益, 随后调整曝光时间和 em 增益, 直到所需的记录条件达到.在大多数情况下, 一个平均信号10-20% 的最大可能像素强度的相机是一个良好的水平目标。一旦达到了适当的记录参数集, 开始纵向获取图像, 如上所述。</li>

5. 灌流组织培养 (可选)
注: 如导言所述, 生物发光培养器适用于非标准组织文化系统的成像。这在发展细胞培养系统中得到了例证。
<ol>
	<li>用口连接器将细胞播种到单通道幻灯片上, 通道深度为0.6 或0.8 毫米, DMEM 有 10% FBS 和笔/链球菌, 如1节所述。如步骤3.2 所述, 制作灌注介质。</li>
	<li>在录音之前, 用1毫米内径 (内径) 准备所需灌注系统的油管ETFE 油管和 1 mm 的硅胶管, 弯头, 男性和女性口配件, 如图 2A所示。 注: 油管长度的大部分是 ETFE 油管, 用2厘米的硅树脂导管将 ETFE 油管连接到口管接头, 随后链接到通道滑轨。</li>
	<li>用70% 乙醇冲洗油管, 然后用无菌 PBS 消毒。</li>
	<li>从孵化器中删除通道幻灯片中的单元格区域性。吸入介质, 用5ml 灌注介质代替。</li>
	<li>用5ml 灌注介质填充20毫升注射器。</li>
	<li>将油管连接到缓冲滑块 (请参见图 2), 但不要将其与含有单元格的幻灯片相连, 并用灌注介质 (3-5 毫升) 冲洗油管和滑动。</li>
	<li>将含细胞的幻灯片连接起来, 再用1毫升的介质冲洗整个系统, 以消除气泡。</li>
	<li>将整个系统转移到生物发光孵化器。</li>
	<li>将介质填充的注射器装入注射器泵, 而不与油管断开连接, 通过整个系统使用泵冲洗1毫升的介质, 以确保系统中没有气泡。</li>
	<li>将泵的直径设置在使用的注射器上。 注: 在这里使用的20毫升注射器, 直径是19.13 毫米。</li>
	<li>将泵的流量设置为50µL/小时, 并开始运行。</li>
	<li>在记录开始前确保任何幻灯片或导管中没有气泡, 因为这些会影响荧光的表达, 当它们穿过细胞单层时。如有必要, 请在整个单元格中刷新进一步的媒体以实现此目的。</li>
	<li>关闭生物发光孵化器门, 并继续在步骤4.7 开始录音。</li>
</ol>
6. 录音过程中的处理
注意: 有时候, 通过记录来治疗细胞是可取的, 无论是药物还是荷尔蒙。在这种情况下, 必须小心处理细胞, 以防细胞振荡在治疗过程中复位。因此, 这是特别重要的是, 细胞保持在一个恒定的温度, 因为这是一个主要的提示为细胞昼夜节律5,6。
<ol>
	<li>在停止记录之前准备好所有的治疗组件。</li>
	<li>准备一个化学等温垫在37° c。</li>
	<li>停止设备记录, 取出待处理的盘子, 放在恒温热垫上。</li>
	<li>按要求对待文化, 并回到生物发光孵化器在相同的位置, 以前。</li>
	<li>重新开始录制。</li>
</ol>
7. 分析
注意: 生物发光孵化器以一系列个人图像的形式生成数据。我们主要使用斐济12来管理这些图像, 然后导出每个区域 (ROI) 的平均像素强度数据以进行进一步分析。
<ol>
	<li>在斐济打开图像栈, 通过单击 "图像" 调整亮度和对比度调整 |亮度/对比度 "和调整滑动条, 直到图像是在一个适当的范围内查看发光信号。</li>
	<li>使用 "分析" 下可用的 ROI 管理器选择感兴趣的领域工具 |ROI 管理器 "。</li>
	<li>通过点击 "ROI 管理器" 导出所选区域的平均信号更多 |Multimeasure "。</li>
	<li>将生成的数据复制到分析软件中。</li>
	<li>手动将数据的时间基准调整为成像的时间间隔 (即、15、30或60分钟间隔, 描述为0.25、0.5 或 1 h), 而不是斐济提供的图像数。 注: 现在可以进行进一步分析, 如确定期间。为此, 下面的等式用于执行非线性回归分析: 
	<img alt="Equation" src="/files/ftp_upload/56623/56623eq1.jpg"> 其中, y是信号, x对应的时间, m是趋势线的渐变, c是趋势线的 y 截距, 振幅是波形上方峰值的高度, k是衰减常数或阻尼率 (例如, 1/k是半衰期), 相位是余弦波中 x 的移位, 周期是整个周期发生的时间13。R2值用作优的指示器。 其他分析方法也是可能的。第一 24 h 的收购应排除在分析, 因为蜂窝 bioluminesence 可能出现瞬态, non-circadian 在这段时间内的变化。这是对媒体变化的敏锐反应的结果。</li>
</ol>

<ol>
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不存在利益冲突。

这里所描述的协议是哺乳动物细胞培养, 无论是在灌注和静态条件。然而, 鳄鱼可以很容易地适应其他模型系统。事实上, 它已经被证明是提供一个优秀的平台, 同时监测运动, 睡眠, 和周边基因表达节律在果蝇在恒定的黑暗中保持15.另据指出, 根据申请的不同, 此处提到的相机类型可能是合适的。我们设想, 使用适当的过滤器, 现有设置的修改版本可以在主体中用于荧光定量。
唯一的应用, 鳄鱼可能不适当的是那些特别高的空间分辨率需要, 如成像的时空组织的 PER2::LUC 表达在器官切片的哺乳动物交叉核, 或其他小组织切片。
鳄鱼使许多实验被执行, 到目前为止不容易地是可实现的由常规录音技术。与目前的生物发光测量方法相比, 鳄鱼在细胞培养皿或滑动片中提供了更大的灵活性, 外部介质条件、灵敏度和 processivity。
这是特别相关的时候, 当有一个远离标准的2D 细胞培养模式, 以3D 化和流培养系统。因此, 预计鳄鱼将提供一种可适应的方法, 在广泛的条件下, 生物荧光可以在许多天和数周内测量。

luciferases 作为基因表达和蛋白质活动的记者, 已成为分子和细胞生物学研究的热门技术。在昼夜节律领域是如此, 萤火虫荧光合成和催化失活的动力学特别适合报告在大约 24 h 昼夜周期内发生的基因表达的纵向变化。因此, 荧光被用来作为一个昼夜记者横跨广泛的有机体, 包括真菌, 植物, 苍蝇和哺乳动物1,2,3,4。
当量化的昼夜节律基因表达在体外, 光电倍增管 (PMT) 通常用于记录生物发光信号。PMT-based 测量的灵活性有限, 但通常被限制在预先确定的盘子或盘子的大小。也不可能从使用 PMT 进行监测的样本中收集任何空间信息, 这可能导致在荧光表达式中显示空间变化的图像样本时丢失信息。此外, 由于 PMT 和相关电子产品在标准细胞培养孵化器的湿化环境中容易发生故障, 因此使用并联的纵向荧光记录总是在 non-humidified 恒温箱中进行。因此, 细胞培养皿必须密封密闭, 以防止蒸发水分流失, 因此培养基必须缓冲 3-(N-吗) 磺酸 (拖把) 或 4-(2-羟乙基)-1-piperazineethanesulfonic 酸 (HEPES), 而不是 CO2/碳酸氢盐缓冲系统, 功能的在体内和日常使用的哺乳动物组织培养。
由于这些限制, 并联的生物荧光测量通常会对实验期间维持细胞的条件施加严格的限制。为了克服这些问题, 也为了增加可能的实验条件的范围, 我们使用了一个标准 CO2/N 个2 170 L 组织培养孵化器, 它已经适应了添加一个 water-chilled 电子倍增电荷耦合器件 (EMCCD) 相机与 anti-mist 光学和数字控制的温度和气体水平。这被称为自动纵向荧光成像气体和温度优化录音机, 或鳄鱼。鳄鱼允许极大地增加生物荧光成像的灵活性, 既用于标准组织培养皿的高通量成像 (同时可达 6 x 96-或 384-井板), 也可用于非标准的组织培养系统, 如作为微流控装置中的灌注细胞。该仪器还允许在湿化条件下进行成像, 并可对 CO2和 O2分压以及温度进行可变控制。
下面的协议描述了一种使用鳄鱼 (从今以后称为 "生物发光孵化器") 的哺乳动物细胞和组织培养系统的生物发光记录方法。然而, 应该指出的是, 该系统将非常适合生物荧光成像, 并在一些修改, 荧光成像, 在一些其他的生物学系统和环境。

<table><tbody><tr><td>DMEM (1x) + GlutaMAX</td><td>Gibco</td><td>31966-021</td><td></td></tr><tr><td>Hyclone FetalClone III Serum</td><td>GE Healthcare</td><td>SH30109.03</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermofisher</td><td>21103049</td><td>basal medium</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A4919</td><td></td></tr><tr><td>Catalase</td><td>Sigma</td><td>C40</td><td></td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G6013</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I1882</td><td></td></tr><tr><td>Superoxide Dismutase</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>Holo-transferrin</td><td>Calbiochem</td><td>616424</td><td></td></tr><tr><td>T3 (triiodo-L-thyronine</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>L-Carnitine</td><td>Sigma</td><td>C7518</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma</td><td>E9508</td><td></td></tr><tr><td>D (+)-Galactose</td><td>Sigma</td><td>G0625</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Sodium Selenite</td><td>Sigma</td><td>S9133</td><td></td></tr><tr><td>Corticosterone</td><td>Sigma</td><td>C2505</td><td></td></tr><tr><td>Linoleic Acid</td><td>Sigma</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma</td><td>L2376</td><td></td></tr><tr><td>Lipoic Acid</td><td>Sigma</td><td>T1395</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Retinol Acetate</td><td>Sigma</td><td>R7882</td><td></td></tr><tr><td>Retinol, all trans</td><td>Sigma</td><td>95144</td><td></td></tr><tr><td>D,L-alpha-Tocopherol</td><td>Sigma</td><td>95240</td><td></td></tr><tr><td>D,L-alpha-Tocopherol acetate</td><td>Sigma</td><td>T3001</td><td></td></tr><tr><td>Sodium Bicarbonate Solution</td><td>Sigma</td><td>S8761-100ML</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Galaxy 170R incubator&amp;nbsp;</td><td>Eppendorf</td><td>CO17301001</td><td></td></tr><tr><td>Luciferin</td><td>Biosynth</td><td>L-8220</td><td></td></tr><tr><td>D -(+)-Glucose solution</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>DMEM powder</td><td>Sigma</td><td>D5030</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>PHG0007</td><td></td></tr><tr><td>1 mm I.D. silicone tubing&amp;nbsp;</td><td>GE Healthcare</td><td>19-4692-01</td><td></td></tr><tr><td>Elbow luer connector</td><td>Ibidi</td><td>10802</td><td></td></tr><tr><td>Male luer fittings</td><td>Ibidi</td><td>10826</td><td></td></tr><tr><td>Female luer fittings</td><td>Ibidi</td><td>10825</td><td></td></tr><tr><td>&amp;micro;-slide luer I 0.6</td><td>Ibidi</td><td>80196</td><td></td></tr><tr><td>BD plastipak 20ml syringe</td><td>Becton Dickinson</td><td>300613</td><td></td></tr><tr><td>1mm I.D. ETFE tubing</td><td>GE Healthcare</td><td>18-1142-38</td><td></td></tr><tr><td>PF670462</td><td>Sigma</td><td>SML0795</td><td></td></tr><tr><td>B27 Supplement (50x)</td><td>ThermoFisher</td><td>17504044</td><td></td></tr><tr><td>iXon Ultra EMCCD camera</td><td>Andor</td><td>iXon 888</td><td></td></tr><tr><td>Fiji</td><td>ImageJ</td><td>N/A</td><td></td></tr><tr><td>Prism 7.0</td><td>Graphpad Software</td><td>N/A</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Deltaphase Isothermal Pad</td><td>Braintree Scientific</td><td>39DP</td><td></td></tr><tr><td>Heated neutral density filter&amp;nbsp;</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>Osmomat 030</td><td>Gonotech</td><td>Discontinued</td><td></td></tr><tr><td>300 mOsmol/kg calibration standard</td><td>Gonotech</td><td>30.9.0020</td><td></td></tr><tr><td>Measuring vessel</td><td>Gonotech</td><td>30.9.0010</td><td></td></tr><tr><td>Focusing cylinder</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>NE-1600 programmable syringe pump</td><td>Pump Systems inc.</td><td>NE-1600</td><td></td></tr><tr><td>Andor Solis Software</td><td>Andor</td><td>N/A</td><td></td></tr></tbody></table>

flexible measurement of bioluminescent reporters using an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (alligator)

Luciferase-based 记者的细胞基因表达是广泛使用的纵向和 end-point 的生物活性测定。例如, 在昼夜节律研究中, 与萤火虫荧光的时钟基因融合, 在细胞生物发光中产生了强健的节律, 持续了许多天。与光电倍增管 (PMT) 或常规 microscopy-based 方法相关的技术限制通常要求在相当 non-physiological 的条件下维持细胞和组织记录, 在灵敏度和吞吐量之间进行权衡。在这里, 我们报告了以前的方法, 允许长期生物荧光成像高灵敏度和吞吐量, 支持广泛的文化条件, 包括可变气体和湿度控制, 并接受许多不同组织培养皿和盘子。这种自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 也允许观察空间变化的荧光表达横跨细胞单层或组织, 这是不能轻易地观察到传统方法.我们强调了鳄鱼是如何提供极大的灵活性, 以检测荧光活动与现有的方法相比。

本文概述了一个协议的生物发光成像的哺乳动物细胞使用鳄鱼 (生物发光孵化器)。这种技术允许在成像生物发光系统的物理设置和细胞外条件的灵活性。本文介绍了简单静态组织培养方法 (图 1、辅助视频 1) 和灌流细胞培养 (图 2、辅助视频 2), 但可以使用该系统对许多其他设置进行映像。所有数据都是使用7节所述的方法进行量化的。
图 1A显示了一个生物发光记录的示例视频, 其中包含 PER2::LUC 成纤维细胞的 6 x 96 井板,4。最外层的井不包含细胞, 因为这些不是这个实验所需要的。细胞经历了不同温度的夹带, 藉以他们接受任一温度周期 12 h, 在32° c 跟随 12 h 37 ° c 为 72 h 或逆 (12 h, 在37° c 跟随 12 h, 在32° c 为 72 h), 在被拿着在恒定的37° c 为录音之前。 每小时服用60分钟。其中的两个条件在图 1B中进行了量化。
图 2A显示了用于灌注组织培养的系统的设置示意图。这包括两个通道幻灯片连接与油管。介质通过注射器泵通过细胞驱动。第一个这些幻灯片的作用是气体渗透气泡陷阱 (缓冲幻灯片) 和不包含细胞, 与第二个包含的细胞, 生物荧光记录。在图 2B中显示了来自此系统的代表录制视频。每15分钟服用15分钟。在这里, 细胞维持在标准灌注条件下或在酪蛋白激酶抑制剂 PF670462 的存在, 这已经显示延长昼夜周期和减少时钟基因表达节律在培养哺乳动物细胞14。对 PER2::LUC 表达式的影响在图 2C (顶部面板) 中显示, 在图 2C (底部面板) 所示的标准静态细胞培养条件下, 使用相同浓度的药物处理的细胞, 具有周期的量化如图 2D所示。从这是清楚的那治疗与 PF670462 影响 PER2::LUC 表示在两套条件之下。然而, 在灌流条件下处理 PF670462 的细胞显示周期延长约 3 h (3 ±0.9 h), 在静态条件下治疗的细胞在药物显示大的时期延长至 #62 期; 48 小时。这可以由一个阻尼余弦, 如7节所述, (extra-sum-of-squares F 测试vs一条直线, p & #60; 0.0001)。有趣的是, 灌注期延长的幅度更接近于观察到的在体内14。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56623/56623fig1v2.jpg"> 
图 1: 数据示例.(A) 示例生物发光孵化器中 6 x 96 井板中永生化 PER2::LUC 生物发光的视频快照。在补充视频 1中用黄色突出显示了要量化的井。(B) 从 A 的生物荧光定量。两组3片细胞采用温度循环 (12 h, 在32° c; 12 h, 在37摄氏度) 相互 anti-phasic, 产生 PER2 蛋白表达的相反阶段 (n = 6, 平均± SEM)。<a href="//ecsource.jove.com/files/ftp_upload/56623/56623fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56623/56623fig2v2.jpg"> 
图 2: 用 CK1δ抑制剂进行灌注.(A) 灌注系统示意图。(B) 示例 PER2::LUC 细胞在灌注下的生物荧光记录的视频快照。量化的样本区域以黄色突出显示。(C) 对 PER2::LUC 成纤维细胞在灌注和静态条件下的生物发光进行量化, 并在无3µM CK1 抑制剂 PF670462 (n = 3, 均值± SEM)。生物发光已经被标准化为最小值和最大数值。(D) 期间分析 (双向方差计算, 栎-Sidak 的多重比较测试)。<a href="//ecsource.jove.com/files/ftp_upload/56623/56623fig2v2large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组件</td>
			<td>最终介质浓度 (μ m)</td>
			<td>库存 (毫克/毫升)</td>
			<td>400毫升 NS21</td>
		</tr>
		<tr>
			<td>牛白蛋白</td>
			<td>37</td>
			<td>-</td>
			<td>50克</td>
		</tr>
		<tr>
			<td>过氧化氢</td>
			<td>0.01</td>
			<td>-</td>
			<td>50毫克</td>
		</tr>
		<tr>
			<td>胱</td>
			<td>3。2</td>
			<td>-</td>
			<td>20毫克</td>
		</tr>
		<tr>
			<td>胰岛素</td>
			<td>0。6</td>
			<td>10</td>
			<td>8毫升</td>
		</tr>
		<tr>
			<td>超氧化物歧化酶</td>
			<td>0.077</td>
			<td>-</td>
			<td>50毫克</td>
		</tr>
		<tr>
			<td>全息-转铁蛋白</td>
			<td>0.062</td>
			<td>-</td>
			<td>100毫克</td>
		</tr>
		<tr>
			<td>T3 (triiodo-甲状腺素)</td>
			<td>0.0026</td>
			<td>2</td>
			<td>20µL</td>
		</tr>
		<tr>
			<td>l-肉碱</td>
			<td>12</td>
			<td>-</td>
			<td>40毫克</td>
		</tr>
		<tr>
			<td>胺</td>
			<td>16</td>
			<td>液体 (1 克/毫升)</td>
			<td>20µL</td>
		</tr>
		<tr>
			<td>d-(+)-半乳糖</td>
			<td>83</td>
			<td>-</td>
			<td>300毫克</td>
		</tr>
		<tr>
			<td>腐</td>
			<td>183</td>
			<td>-</td>
			<td>322毫克</td>
		</tr>
		<tr>
			<td>亚硒酸钠</td>
			<td>0.083</td>
			<td>1</td>
			<td>280µL</td>
		</tr>
		<tr>
			<td>皮质</td>
			<td>0.058</td>
			<td>2</td>
			<td>0.2 毫升</td>
		</tr>
		<tr>
			<td>亚油酸</td>
			<td>3。5</td>
			<td>100</td>
			<td>0.2 毫升</td>
		</tr>
		<tr>
			<td>亚麻酸</td>
			<td>3。5</td>
			<td>100</td>
			<td>0.2 毫升</td>
		</tr>
		<tr>
			<td>硫辛酸</td>
			<td>0。2</td>
			<td>4。7</td>
			<td>0.2 毫升</td>
		</tr>
		<tr>
			<td>孕</td>
			<td>0.02</td>
			<td>3。2</td>
			<td>0.04 毫升</td>
		</tr>
		<tr>
			<td>醋酸黄醇</td>
			<td>0。2</td>
			<td>20</td>
			<td>0.1 毫升</td>
		</tr>
		<tr>
			<td>视黄醇, 所有反式</td>
			<td>0。3</td>
			<td>10</td>
			<td>0.2 毫升</td>
		</tr>
		<tr>
			<td>D, l-α-生育酚</td>
			<td>2。3</td>
			<td>100</td>
			<td>0. 2 毫升</td>
		</tr>
		<tr>
			<td>D, l-α-生育酚醋酸酯</td>
			<td>2。1</td>
			<td>100</td>
			<td>0.2 毫升</td>
		</tr>
	</tbody>
</table>
表 1: NS21 准备。
补充视频 1: 来自井板的生物发光的示例视频。例子视频快照的生物发光从永生化 PER2::LUC 在 6 x 96 井板在生物发光孵化器。要量化的水井用黄色突出显示。<a href="http://ecsource.jove.com/files/ftp_upload/56623/Video_1A_Revision-1.avi">请单击此处下载此文件.</a>
补充视频 2: 在灌注下 PER2::LUC 细胞的生物发光记录的示例视频快照.量化的样本区域以黄色突出显示。<a href="http://ecsource.jove.com/files/ftp_upload/56623/Video_2B_Revision-1.avi">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

基因编码荧光是一种受欢迎的非侵入性的转基因记者。使用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼), 使纵向记录从生物发光细胞在广泛的条件。在这里, 我们展示了如何在生理节律研究的背景下使用鳄鱼。

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

flexible-measurement-bioluminescent-reporters-using-an-automated

Nanyang Technological University

Research

JoVE Journal

Biology

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

7.8K 次观看.  MRC Laboratory of Molecular Biology. 基因编码荧光是一种受欢迎的非侵入性的转基因记者。使用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼), 使纵向记录从生物发光细胞在广泛的条件。在这里, 我们展示了如何在生理节律研究的背景下使用鳄鱼。

视频: Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

建立颅内脑肿瘤异种移植肿瘤的生长和对治疗的反应与后续利用生物发光成像分析

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

科研

神经科学

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

免疫与感染

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

医学

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

生物学

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

发育生物学

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

优化人肠道类肠生物用于昼夜节律研究

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

作者聚焦：多细胞系统中昼夜节律的体外研究

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

使用类器官中的生物发光报告基因监测昼夜节律振荡

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...