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 border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM (1x) + GlutaMAX</td>
			<td style="width:145px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hyclone FetalClone III Serum</td>
			<td style="width:145px;">GE Healthcare</td>
			<td>SH30109.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
			<td style="width:145px;">Thermofisher</td>
			<td>21103049</td>
			<td>basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:145px;">Sigma</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Catalase</td>
			<td style="width:145px;">Sigma</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutathione</td>
			<td style="width:145px;">Sigma</td>
			<td>G6013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:145px;">Sigma</td>
			<td>I1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superoxide Dismutase</td>
			<td style="width:145px;">Sigma</td>
			<td>S5395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Holo-transferrin</td>
			<td style="width:145px;">Calbiochem</td>
			<td>616424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T3 (triiodo-L-thyronine</td>
			<td style="width:145px;">Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Carnitine</td>
			<td style="width:145px;">Sigma</td>
			<td>C7518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanolamine</td>
			<td style="width:145px;">Sigma</td>
			<td>E9508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D (+)-Galactose</td>
			<td style="width:145px;">Sigma</td>
			<td>G0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Putrescine</td>
			<td style="width:145px;">Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Selenite</td>
			<td style="width:145px;">Sigma</td>
			<td>S9133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Corticosterone</td>
			<td style="width:145px;">Sigma</td>
			<td>C2505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linoleic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>L1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linolenic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>L2376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipoic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>T1395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Progesterone</td>
			<td style="width:145px;">Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retinol Acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>R7882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retinol, all trans</td>
			<td style="width:145px;">Sigma</td>
			<td>95144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D,L-alpha-Tocopherol</td>
			<td style="width:145px;">Sigma</td>
			<td>95240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D,L-alpha-Tocopherol acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>T3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate Solution</td>
			<td style="width:145px;">Sigma</td>
			<td>S8761-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GlutaMAX (100x)</td>
			<td style="width:145px;">Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:145px;">Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galaxy 170R incubator&#160;</td>
			<td style="width:145px;">Eppendorf</td>
			<td style="width:119px;">CO17301001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin</td>
			<td style="width:145px;">Biosynth</td>
			<td style="width:119px;">L-8220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D -(+)-Glucose solution</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">G8644-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">D5030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MOPS</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">PHG0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mm I.D. silicone tubing&#160;</td>
			<td style="width:145px;">GE Healthcare</td>
			<td>19-4692-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Elbow luer connector</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Male luer fittings</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Female luer fittings</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#181;-slide luer I 0.6</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">80196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD plastipak 20ml syringe</td>
			<td style="width:145px;">Becton Dickinson</td>
			<td style="width:119px;">300613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1mm I.D. ETFE tubing</td>
			<td style="width:145px;">GE Healthcare</td>
			<td style="width:119px;">18-1142-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PF670462</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">SML0795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B27 Supplement (50x)</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iXon Ultra EMCCD camera</td>
			<td style="width:145px;">Andor</td>
			<td style="width:119px;">iXon 888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiji</td>
			<td style="width:145px;">ImageJ</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Prism 7.0</td>
			<td style="width:145px;">Graphpad Software</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deltaphase Isothermal Pad</td>
			<td style="width:145px;">Braintree Scientific</td>
			<td style="width:119px;">39DP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated neutral density filter&#160;</td>
			<td style="width:145px;">Cairn Research</td>
			<td style="width:119px;">Custom item</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmomat 030</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">Discontinued</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">300 mOsmol/kg calibration standard</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">30.9.0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Measuring vessel</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">30.9.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Focusing cylinder</td>
			<td style="width:145px;">Cairn Research</td>
			<td style="width:119px;">Custom item</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NE-1600 programmable syringe pump</td>
			<td style="width:145px;">Pump Systems inc.</td>
			<td style="width:119px;">NE-1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor Solis Software</td>
			<td style="width:145px;">Andor</td>
			<td style="width:119px;">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

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

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

使用IVIS频谱的乳腺肿瘤在体内生物发光成像

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

科研

生物学

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

斑马鱼化工屏幕自动成像和分析的发展。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

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 ...

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)7. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices8. To date, leaf shape programs (e.g. SHAPE9, LAMINA10, LeafAnalyzer11, LEAFPROCESSOR12) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size13. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.

LeafJ:ImageJ的插件半自动叶形状测量

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

细胞外离子流的使用离子选择性自引用的微电极技术测量

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

三维球体制作一个高效，灵活细胞聚集方法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

使用自动成像系统测量 iPSC 的汇合度。

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...