我们感谢阿拉文森·塞缪尔博士和马克·格肖博士帮助开发了行为装置的初始版本，马修·巴蒂博士修改了行为装置。这项研究得到了辛辛那提儿童医院、JST/PRESTO、迪姆斯和NIH R01 GM107582到F.N.H.的受托人格兰特的支持。

1. 准备苍蝇
<ol><li>浅暗（LD）实验
	<ol>
<li>在光12小时/暗12小时（LD）周期下，在孵化器中提高苍蝇（25°C/40-60%相对湿度（RH））。孵化器的光强度为 500-1，000 勒克斯。</li>
		<li>在 24 小时内完成行为检测需要两个孵化器。两个孵化器都应具有具有开机功能的可编程光。它们还应有无法透射到光的实心门（即 没有玻璃或玻璃杯）。 注意:一个孵化器应指定为"日"孵化器，并设置为 LD 周期，12 小时光和 12 小时黑暗。第二个孵化器应指定为"夜间"孵化器，并设置为第一个孵化器的反向，12小时黑暗，然后是12小时光。夜间孵化器必须放置在黑暗中可访问的房间里，这样，体验夜间条件的苍蝇就可以轻松地进入实验。</li>
		<li>将飞瓶放在白天或晚上的孵化器中。收集新孵化的苍蝇在一个新的小瓶，20-30每检测，并保持在同一孵化器2-3天。</li>
		<li>2-3天后，使用苍蝇进行温度偏好行为检测。<ol>
<li>对于日（泽格伯时间 （ZT） 0-12） 实验，从日孵化器收集苍蝇。</li>
			<li>就在行为实验之前，将收集的飞瓶带出日间孵化器。</li>
			<li>对于夜间（ZT 13-24）实验，从夜间孵化器收集苍蝇。</li>
			<li>就在行为实验之前，将收集的飞瓶带出夜间孵化器，用铝箔包裹，放在暗室的一个盒子里，放在红灯下。 注意:由于夜间实验的温度偏好行为检测是在黑暗中进行的，所以必须防止对苍蝇的光线暴露，直到行为实验结束。 注意:在实验进行当天，苍蝇不应接触到二氧化碳。</li>
		</ol>
</li>
	</ol></li>
	<li>恒定黑暗 （DD） 实验
	<ol>
<li>
DD日
		<ol>
<li>DD 日实验需要额外的孵化器，我们称之为手稿其余部分的"过渡"孵化器。过渡孵化器必须放置在黑暗中可访问的房间里，这样，体验DD条件的苍蝇就可以轻松访问进行实验。过渡孵化器的示例灯时间表将在下午 1 点到 7 点开灯，晚上 7 点到 1 点（图 1）关闭。</li>
			<li>收集在日孵化器中饲养的苍蝇。当灯亮起时，在下午 1 点到 7 点之间将飞瓶放在过渡孵化器中。这样，苍蝇被正确地暴露在光线下，直到晚上7点，此时光线关闭。</li>
			<li>第二天下午1点前，在黑暗条件下，将飞瓶从过渡孵化器中取出，用铝箔包裹，放在一个盒子里。将盒子放在任何孵化器中再放一天。</li>
		</ol>
</li>
		<li>
DD之夜
		<ol>
<li>收集在暗黑中在夜孵化器中升起的苍蝇，在红灯下。或在夜间孵化器的灯光亮起时额外收集苍蝇。</li>
			<li>只要灯熄灭，在黑暗中用铝箔包裹收集的小瓶，并将小瓶放在一个盒子里。将盒子放在任何孵化器中再放两天（图1B）。 注意:由于夜间实验的温度偏好行为检测是在黑暗中进行的，所以必须防止对苍蝇的光线暴露，直到行为实验结束。</li>
		</ol>
</li>
	</ol></li>
	<li>恒光（LL）实验
	<ol>
<li>
LL 日
		<ol>
<li>LL日实验需要额外的孵化器。此孵化器保持LL条件（25°C，800勒克斯），并持续开灯。</li>
			<li>收集在日培养箱中饲养的苍蝇。在其"白天"随时将飞瓶放在LL孵化器中。</li>
		</ol>
</li>
		<li>
LL 之夜
		<ol>
<li>收集在夜间孵化器中饲养的苍蝇。在夜间孵化器的灯光打开期间，将飞瓶从夜间孵化器转移到LL孵化器。 注意:例如，夜间孵化器中的灯在上午 7 点关闭。早上 7 点前将飞瓶从夜间孵化器转移到 LL 孵化器，并将小瓶在 LL 孵化器中再保存 4 天。 注:在LL条件下的第4天，运动活动的振荡被废除15，16，而TPR仍然持续3。</li>
		</ol>
</li>
	</ol></li>
</ol>2. 温度偏好行为测定装置
<ol><li>将玻璃杯盖（29 厘米 x 19.2 厘米）（图 4）放在铝板上。</li>
	<li>监测板与盖之间的温度。六个温度探针在一条车道内盖内的不同位置连接（图2）。 注意:确保探针不接触铝板或玻璃杯盖。气温应设置为 18-32 °C 的梯度。</li>
	<li>将设备放置在维持在 25 °C/65-75% RH 的环境室中。这个房间需要被封锁，远离任何外部光线。环境室通常配有风扇，以保持一定的温度和湿度。 注意:风扇的空气可能会干扰仪器上稳定的温度梯度。为了防止这种情况，我们使用透明纸张，覆盖仪器周围的区域。</li>
	<li>准备温度计和催眠仪，检查环境室的温度和湿度。 注意:光影响 嗜血杆菌3的温度偏好。设备上应均匀提供相同强度的光。我们的环境室灯的强度是~800勒克斯。 注意:完成行为实验后，将从 CO2 罐连接的管子或电源放在仪器顶部孔附近，以便麻醉和清除苍蝇。</li>
</ol>3. 准备使用设备
<ol><li>打开仪器至少30分钟，以便正确确定板表面的温度梯度（图3）。</li>
	<li>将行为装置的盖子涂上防水剂，以防止苍蝇攀爬盖子的墙壁或天花板。擦去多余的防水剂，将盖子保持25-30分钟干燥。</li>
	<li>去除铝板上的任何凝结物。将玻璃杯盖放在铝板上，用六个 C 夹子固定（图 2）。 注意:如果有必要，盖子密封良好是非常重要的。</li>
	<li>将盖子保留至少 15 分钟。铝板和盖子之间的空气温度梯度为 18-32 °C。</li>
</ol>4. 温度偏好行为测定
<ol><li>通过盖子每个车道中心的小孔（图 2 和 4）将苍蝇装载到铝板和仪器的玻璃盖之间的空间。用盖子遮盖孔，防止苍蝇逃跑。</li>
	<li>对于黑暗的环境，关掉环境室的所有灯。当苍蝇被放入仪器时，可以使用红灯。在行为实验完成之前，确保苍蝇不会暴露在除红灯以外的任何灯光下。</li>
	<li>对于每次试验，使用20-25只成年苍蝇，这些苍蝇不会在随后的试验中重复使用。行为检测执行 30 分钟。拍几张照片有或没有闪光灯。在实验过程中，要小心不要发出太大的噪音或任何突然的动作。</li>
	<li>记录仪器上所有六个探针的温度。注意室温和湿度。</li>
	<li>用二氧化碳气体麻醉仪器中的苍蝇，松开夹子，取下玻璃杯盖，将苍蝇从盘子中取出。每次实验后，苍蝇都会被丢弃。擦去盘子里的任何凝结物或水分。更换板上的盖子，用夹子拧紧，为下一个实验做准备。</li>
	<li>为了代表全天的温度偏好，24 小时时段分为 8 个时区，白天 4 个时区，晚上 4 个时区。例如，我们使用这些 ZT 或 CT 1-3、4-6、7-9、10-12、13-15、16-18、19-21 和 22-24。 注意:由于遮蔽效果引起的表型变化预计在开启 （ZT0） 或关闭 （ZT12） 之后立即发生，我们不检查这些时间的温度偏好行为（ZT 或 CT 0-1、11.5-13 和 23.5-24）。每个时区必须至少进行五次试验，以便结果在统计学上是合理的。</li>
</ol>5. 数据分析
<ol><li>计算温度梯度如下:根据放置在沿边缘的玻璃盖顶部和底部的两个尺子（图 2A）确定温度探针的位置。</li>
	<li>温度探针之间的温度梯度估计是线性的。根据温度探头的位置及其相应的记录温度，在图片的适当位置绘制代表每个温度的线条。计算位于每个度区间的苍蝇数量。不包括墙壁上的任何苍蝇或盖子的天花板。</li>
	<li>计算每个车道每个温度范围内的苍蝇百分比。平均首选温度是通过计算每个间隔的苍蝇和温度百分比的产品计算的，如下所示: 百分比 的苍蝇 x 18.5 °C = 百分比的苍蝇 x 19.5 °C = 百分比的苍蝇 x 20.5 °C 。。</li>
	<li>计算每个时区的平均首选温度:每个时区（ZT 1-3、4-6、7-9、10-12、13-15、16-18、19-21 和 22-24）执行温度偏好行为>5 次。要计算每个时区的平均首选温度，每个试验的平均首选温度一起平均。s.e..m错误栏等于试验之间的错误。</li>
</ol>

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没有什么可透露的。

在这里，我们说明了温度偏好行为装置的细节和TPR行为分析。 德罗索菲拉 展示了时钟控制的 TPR 的突出、坚固和可重复的功能。然而，我们的数据表明，至少有两个因素，环境光和年龄，严重扰乱TPR行为表型。
我们观察到，光显著影响 德罗索菲拉的温度偏好。这与在LD中保存 的1118 只苍蝇比DD中的苍蝇更喜欢白天的温度更高，尽管在LD和DD3</sup...

温度是一种无处不在的环境线索。动物表现出各种行为，以避免有害的温度和寻求舒适的。 嗜血杆菌 表现出一个强大的温度偏好行为6，7。当苍蝇被释放到温度梯度从18-32 °C，苍蝇避免温暖和寒冷的温度，最后选择在3日早晨25°C的首选温度。暖温传感器是一组热感官神经元，交流神经元，它表达 嗜血杆菌 瞬态受体电位（TPR）通道，TRPA16，9。冷温传感器位于第三天线段，因为消融第三个天线段会导致缺乏冷温避免6。最近，TRPP蛋白布里维多（Brv）被确定为10。由于 Brv 在第三个天线段中表示并调解冷检测，Brv 是一种可能的冷感应分子，对温度偏好行为至关重要。总之，苍蝇使用这两个温度传感器来避免温暖和寒冷的温度，并找到一个首选的温度。
虽然哺乳动物产生热量来调节体温，但息热通常使体温适应环境温度11。众所周知，一些教会每天都会表现出一种TPR行为，这被认为是教会调节BTR12的策略。为了确定苍蝇是否表现出TPR，我们在24小时内重复了不同点的温度偏好行为分析。我们发现， 德罗索菲拉 每天表现出TPR，这是在早上低，在晚上高，并遵循类似于BTR在人类13的模式。
在 德罗索菲拉，大脑中有大约150个时钟神经元。调节运动活动的时钟神经元称为M和E振荡器。然而，有趣的是，M和E振荡器不调节TPR，相反，我们表明，DN2时钟神经元在大脑中调节TPR，但不是运动活动。这些数据表明，TPR独立于运动活动进行调节。值得注意的是，哺乳动物BTR也独立于运动活动调节。大鼠的消融研究表明，BTR通过特定的SCN神经元进行控制，这些神经元针对的是与控制运动活动14的神经元不同的亚参数区神经元。因此，我们的数据考虑了哺乳动物BTR和苍蝇TPR进化保存的可能性3，因为飞TPR和哺乳动物BTR都表现出昼夜时钟依赖温度的节奏，这是独立于运动运动活动的调节。
在这里，我们描述了如何分析 德罗索菲拉的TPR行为检测的细节。这种方法不仅可以研究TPR的分子机制和神经回路，还可以研究大脑如何整合不同的环境线索和内部生物钟。

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			<td height="41" style="height:41px;width:172px;">Bright Lab Jr. Safelight</td>
			<td style="width:109px;">Amazon</td>
			<td style="width:153px;">#B00013J8UY</td>
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			<td style="width:153px;">TR67-32</td>
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			<td height="52" style="height:52px;width:172px;">Generic 480 Watt ATX power supply</td>
			<td style="width:109px;"></td>
			<td style="width:153px;"></td>
			<td style="width:144px;">computer cooling system</td>
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			<td height="64" style="height:64px;width:172px;">MCR220-QP-RES Dual 120 mm Radiator with reservoir&#160;</td>
			<td style="width:109px;">Swiftech</td>
			<td style="width:153px;"></td>
			<td style="width:144px;">computer cooling system</td>
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		<tr height="50">
			<td height="50" style="height:50px;width:172px;">MCP350 In-Line 12V DC pump</td>
			<td style="width:109px;">Swiftech</td>
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			<td height="49" style="height:49px;width:172px;">MCW50 graphics Card liquid cooler</td>
			<td style="width:109px;">Swiftech</td>
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			<td height="52" style="height:52px;width:172px;">Scythe Kaze-Jyuni SY1225SL12SH fan</td>
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</table>

design and analysis of temperature preference behavior and its circadian rhythm in drosophila

昼夜钟调节生活的许多方面，包括睡眠，运动活动，和体温（BTR）节律1，2。我们最近发现了一种新的环食素输出，称为温度偏好节奏（TPR），其中苍蝇的首选温度在白天上升，在夜间下降3。令人惊讶的是，TPR和运动活性通过不同的昼夜神经元3控制。恐龙运动活动是一种众所周知的昼夜行为输出，为发现许多保存的哺乳动物昼夜钟基因和机制4提供了强有力的贡献。因此，了解TPR将导致迄今未知的分子和细胞昼夜机制的识别。在这里，我们描述如何执行和分析 TPR 分析。这项技术不仅能够解剖TPR的分子和神经机制，而且为整合不同环境信号和调节动物行为的大脑功能的基本机制提供了新的见解。此外，我们最近公布的数据表明，苍蝇TPR与哺乳动物BTR3共享功能。嗜血杆菌是息息的，其中通常调节体温的行为。因此，TPR是一种策略，用于在这些苍蝇5-8产生有节奏的体温。我们相信，进一步探索果蝇TPR将有助于描述动物体温控制机制的特征。

温度偏好节奏的示例显示在图5 中。如果行为程序成功，苍蝇应表现出TPR，在TPR中，它们更喜欢在早晨的低温和在晚上的较高温度。无论遗传背景如何，白天温度偏好的+1-1.5 °C增加应在白天观察到，因为我们显示，w1118、yw和广州 S 苍蝇在白天3中表现出类似的温度偏好。
<img alt="Figure 1" fo:content-width=...

Watch this Scientific Journal Video about Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila at JoVE.com

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

我们最近发现了一种新的 环食 素输出，温度偏好节奏（TPR），其中苍蝇的首选温度在白天上升，在夜间下降。TPR 独立于另一个昼夜输出（运动活动）进行调节。在这里，我们描述了在 德罗索菲拉TPR的设计和分析。

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

design-analysis-temperature-preference-behavior-its-circadian-rhythm

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Cell Biology

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Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

Unit 1

Alternative Signaling Routes in Gene Expression

SCIENTISTS IN ACTION

8.0K 次观看.  Cincinnati Childrens Hospital Medical Center. 我们最近发现了一种新的 环食 素输出，温度偏好节奏（TPR），其中苍蝇的首选温度在白天上升，在夜间下降。TPR 独立于另一个昼夜输出（运动活动）进行调节。在这里，我们描述了在 德罗索菲拉TPR的设计和分析。

视频: Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

高分辨率测量的气味驱动在果蝇幼虫的行为

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

科研

生物学

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡<em&gt;果蝇</em&gt;合胞胚胎

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

提取与分析皮质醇从人与猴的毛

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子发生的细胞学分析:Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

观察和交配行为的量化在我国松材线虫，<em&gt;松材线虫</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

不同光照条件下的拟南芥生长行为分析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

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