We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

所有涉及动物的程序进行根据西北大学机构动物护理和使用委员会（IACUC）批准的指导进行。 1.鼠标型号<ol><li>使用8-12周龄小鼠的运动训练。以检测体内行使诱导自噬，用于成像研究和C57BL / 6小鼠进行生化分析的GFP-LC3转基因小鼠（C57BL / 6背景）。 </li></ol> 2.运动诱发的自噬<ol><li> 跑步机设置-强制运动 <ol><li>适应和培养在10°上坡开跑步机小鼠2天。在第1天，锻炼小鼠5分钟以8米/分，并在第2天，运动5分钟将小鼠在8米/分钟，随后另外5分钟，在10米/分钟10。鼓励小鼠温柔的手轻推运行。 </li><li>在第 3天，让小鼠经历对10°上坡跑步机，starti运行90分钟的单一回合以10米/分钟的速度纳克。 40分钟后，增加跑步机的速度以分钟1米/分钟，每10的速度，总共30分钟，然后增加的速度的速度1m /分钟，每5分钟，直到其达到17，米/分钟，总共的运动时间90分钟1070米转动的距离。 </li><li>在低强度范围（1.2赫兹）设置电刺激，鼓励老鼠跑，以避免重复一脚惊世骇俗。 </li><li>对于组织收集，通过安乐死吸入异氟醚小鼠，颈椎脱位运动后即刻。其他核准安乐死方法也可以使用，如CO 2或其他麻醉剂的IP注射。自噬的水平似乎没有由安乐死方法的影响。 </li></ol></li><li> 跑轮设置-自由运动 <ol><li>放置一个笼子内的鼠标运行轮（11.4厘米直径的）用单容纳鼠标。 </li><li>验证使用自行车里程表的运行能力。成立[每车轮的完全旋转距离（毫米）]车轮参数在358（11.4 *π）。测量后24小时运行距离。 </li><li>让鼠标自愿运行2周运行的轮子。对于组织收集，牺牲在上午鼠标的运行期。 </li></ol></li></ol> 3.自噬通量评价<ol><li>为了测量静止和运动的条件下的自噬通量，治疗的小鼠与自噬抑制剂氯喹三天比较它们用PBS处理的小鼠。 <ol><li>溶解氯喹在PBS（5毫克/毫升），以及腹膜内的剂量为50mg / kg /天的连续三天注射喹到小鼠。牺牲小鼠和最后一次注射后收集组织3小时。 </li><li>为行使小鼠以50mg / kg /天的剂量与氯喹预处理他们连续两天。在第三天，注入小鼠与氯喹的相同浓度，和后90分钟让他们在跑步机上另外90分钟运行。 </li><li>安乐死通过异氟烷吸入将小鼠，随后通过颈椎脱位。收集组织运行后，使氯的总温育时间是一样的对照（静止）小鼠（3小时）。 注:另外，用氯喹单次注射，以确定在小鼠组织中自噬通量。 </li></ol></li></ol> 4.组织收获和固定<ol><li>在7.4的最终pH在PBS:在此之前组织准备收获的磷酸盐缓冲液（PBS）和新鲜的4％多聚甲醛（必要的个人防护装备PFA，慎用）。储存在4℃下两种解决方案。 </li><li>填写30毫升注射器15 - 20毫升4％PFA的（对于GFP-LC3的小鼠）。连接到一个20G的针头导管注射器。 </li><li>的运动后，通过异氟烷吸入安乐死的小鼠，随后通过颈椎脱位。 </li><li>将动物上的BA清洁的工作面上CK。剪切和通过切割膜片暴露心脏打开胸腔。 </li><li>作一小切口对肝脏，让血出来，缓缓注入一共有15毫升4-％PFA进入经导管心脏的右心室，直到肺和肝脏变成完全苍白。立即灌注鼠标运动后，使用注射泵以90毫升/小时的恒定流速。 </li><li>灌注后，取出针，并收集感兴趣的组织（ 如大脑11和骨骼肌）。对于骨骼肌，解剖股外侧。简要地说，拉腿的皮肤向后暴露的肌肉，本地化股四头肌12和剖析出股外侧肌，这是外部肌肉附连到股骨的上部。 </li><li>为进一步固定和脱水，放置的GFP-LC3的小鼠的组织在4％PFA 24小时，在4℃下，然后将它们转移到15％的蔗糖的PBS在4℃过夜，或直到组织定居，然后至30％蔗糖的PBS在4℃下过夜或更长的储存空间。保持在黑暗中尽可能样品，因为它们可能对强光敏感。 </li><li>对于免疫印迹分析单元从直接C57BL / 6小鼠在液氮冷冻组织，并将其存储在-80℃。 </li></ol> 5. GFP-LC3泪点的影像学分析<ol><li> 组织流程 <ol><li>预治疗在包埋介质中的组织（预先固定在PFA和存储在30％的蔗糖），例如用于在标记的小培养皿几分钟"最佳切削温度的化合物"（OCT）。 </li><li>在含有足够新鲜OCT以覆盖组织的标记cryomold转移组织。东方朝向cryomold底部的切片表面（对于股截面外侧肌和矢状切面为半大脑）。避免产生气泡。 </li><li> FRE通过将cryomold在填充有干冰带盖泡沫冷却器几分钟EZE样品，直到在OCT变为白色。裹个体样品中的标记的薄片，它们密封在塑料袋中，并把它们存储在-80℃下过夜或更长时间。 </li><li>使用低温恒温器在厚度为10微米的切割样品部分，并且安装在幻灯片的节。存储载玻片在-20℃，并总是从光保持滑动距离只要有可能。 </li></ol></li><li> 幻灯片准备 <ol><li>从冰箱中取出载玻片并在室温下解冻它们。覆盖带安装媒体用DAPI一滴的组织切片。在上面放置适当大小的盖玻片，并避免形成气泡。 </li><li>用指甲油密封盖玻片的边缘，允许指甲油在室温下黑暗中干燥，然后在一个不透光的容器中的载玻片储存在4℃下，或用GFP-LC3泪点的成像捕获进行。 </li></ol></li><li> &lt;GFP的STRONG&gt;量化 - 通过荧光显微镜LC3泪点<ol><li>执行荧光显微镜，捕捉在相同条件下对所有样品的照片（曝光和设置）。的GFP，使用525纳米的发射波长;对于DAPI，使用490纳米。获得每使用股外侧一个60X物镜和大脑的额脑皮层区样品至少十个图像。 </li><li>量化在2500微米2进行统计分析每个图像中的组织区域，失明的实验组的GFP-LC3泪点的数目。计算从各样品的10个图像的平均数目，用"对象计数"的自动测量功能。对于股设置外侧肌是:阈值L = 10，H = 200; 2X流畅; 1X干净，并为脑额叶皮层设置:阈值L = 11，H = 200; 2X流畅; 1X干净。 </li></ol></li></ol> 6. Western印迹分析自噬标记<ol><li> &lt;STRONG&gt;组织过程<ol><li>制备裂解缓冲液:50mM的Tris-HCl; 150毫摩尔NaCl; 1毫摩尔EDTA; 1％的Triton X-100;蛋白酶抑制剂和磷酸酶抑制剂（新鲜添加）。 </li><li> -80℃储存除去C57BL / 6小鼠的组织。切开肌肉组织切成小块之前用以下方法刀片。添加800微升（对于半脑）或500微升（对于股外侧）冷的裂解缓冲液对样品的。与中速均化器均质化，在4℃下的样品。 </li><li>在4℃下用旋转另外的30分钟的均化的混合物充分溶解到1小时。 </li><li>降速匀浆在12,000×g离心10分钟，在4℃，弃沉淀，并回收上清在新管中。 </li></ol></li><li> 自噬分析 <ol><li>确定根据制造商的说明使用BCA蛋白测定试剂盒组织裂解液的蛋白质浓度。规范化每个样品以相同的浓度。 </li><li>在1与2×Laemmli样品缓冲液合并样品:1的比例，煮沸，在95℃，5 - 10分钟，并与自噬免疫印迹分析标记物13，14进行，例如P62（抗p62的抗体， 1:500稀释）与LC3（抗LC3抗体，1:500稀释度）。煮沸样品加入样品缓冲液后立即，如在冰上再温育可以生成LC3的多个退化频带。 </li><li>量化使用ImageJ通过光密度分析p62蛋白和LC3频段，并且归一化值到相应肌动蛋白条带。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

自噬是分解代谢过程提供能量和细胞质组件或受损细胞器溶酶体降解减少细胞毒性。自噬研究重要的是要了解细胞稳态的调节和应激反应的机制。新的模式和方法不断涌现的研究领域15，研究如何吞噬受损有助于众多病理过程16,17。 营养缺乏（饥饿）和药理学诱导常用的方法来诱导自噬在体外和体?...

自噬是一种进化上保守的降解途径，这是响应于各种胁迫条件如饥饿，缺氧1,2诱导。在自噬，双膜囊，称为自噬体，包括不必要的或损坏的亚细胞成分，并将它们运送到溶酶体降解3。基底自噬是在许多疾病，包括神经变性，肿瘤和2型糖尿病4，5，6被牵连于细胞功能和生物体的发展，和受损基底自噬是必不可少的。 最知名的生理自噬诱导剂是饥饿。然而，它有两个主要的限制。首先，饥饿需要很长的时间，以有效地诱导自噬在动物中， 例如 ，在小鼠中食物限制的48小时在大多数器官。第二，饥饿勉强诱导脑自噬，由于大脑中的相对稳定的营养供给。实际上，它也难以通过小分子诱导剂来检测自噬诱导，因为许多药物不能通过血脑屏障。因此，为了更好地分析自噬活化在疾病的发病机制的功能，我们最近发现，运动是诱导自噬在时间7，8，9短时间内更有效的生理方法。与饥饿相比，自噬是有效地跑步机上跑步一样快，30分钟所致。因此，运动是一种方便和有效的生理方法来研究细胞自噬机制在调解医疗福利和预防疾病。 有用于检测自噬活动的，包括LC3和p62的几种蛋白质的标志物。 LC3（微管相关蛋白1A / 1B-光速c海恩3）是胞质蛋白（LC3-I形式）是，在自噬诱导缀合的PE（磷脂酰乙醇胺）。 PE-脂化LC3（LC3-II的形式）被募集到autophagosomal膜，可用于当用GFP标记的可视化自噬。其从细胞溶质向点状在显微镜下自噬体结构易位是自噬诱导的指示。 P62是自噬底物（如泛素化蛋白质）货物的受体，并且被并入自噬体为好。因为蛋白质是与自噬体沿溶酶体降解，其水平可用于测量自噬通量。在这里，我们展示了如何使用这些标志通过有氧运动，包括强迫运动（跑步机）和自愿运动（跑步轮）引起不同的鼠标组织量化自噬。相同的过程也可以治疗其它诱导物后施加到自噬的体内测量。

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			<td height="21" style="height:21px;width:247px;">Treadmill</td>
			<td style="width:204px;">Columbus Instruments</td>
			<td style="width:195px;">150-RM Exer 3/6</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mouse running wheel</td>
			<td style="width:204px;">Super Pet</td>
			<td style="width:195px;">100079365</td>
			<td>diameter 11.4 cm</td>
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			<td height="21" style="height:21px;width:247px;">Odometer</td>
			<td style="width:204px;">Bell</td>
			<td style="width:195px;">DASHBOARD 100</td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">Syringe pump</td>
			<td style="width:204px;">KD Scientific</td>
			<td style="width:195px;">KDS100</td>
			<td></td>
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			<td height="42" style="height:42px;width:247px;">Fluorescence microscope</td>
			<td style="width:204px;">Nikon</td>
			<td style="width:195px;">Model: inverted microscope ECLIPSE</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:247px;">Cryostat</td>
			<td style="width:204px;">Leica</td>
			<td style="width:195px;">CM 1850UV</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Homogenizer</td>
			<td style="width:204px;">IKA</td>
			<td style="width:195px;">003737001 / Model: T10 Basic S1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Chloroquine</td>
			<td style="width:204px;">CAYMAN CHEMICAL COMPANY</td>
			<td style="width:195px;">14194</td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">Parafolmaldehye</td>
			<td style="width:204px;">SIGMA-ALDRICH</td>
			<td style="width:195px;">P6148</td>
			<td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mounting media</td>
			<td style="width:204px;">Vector Laboratories</td>
			<td style="width:195px;">H-1200</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">p62 antibody</td>
			<td style="width:204px;">BD Biosciences</td>
			<td style="width:195px;">610833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LC3 antibody</td>
			<td style="width:204px;">Novus Biologicals</td>
			<td style="width:195px;">NB100-2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X Laemmli Sample Buffer</td>
			<td style="width:204px;">Bio-Rad Laboratories</td>
			<td style="width:195px;">161-0737</td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">ImageJ</td>
			<td style="width:204px;">NIH</td>
			<td style="width:195px;"></td>
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activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

本协议描述两种不同的方法来诱导自噬通过有氧运动小鼠组织:共90分钟由驯化两天进行一个多车道的跑步机锻炼强迫的;两个星期自愿行使对单只装一个用来跑轮。在每个运动方案，我们可以通过测量各个器官荧光显微镜和Western blot分析自噬通量。 我们用表达GFP标记的LC3当记者通过系统的锻炼1，监测自噬的转...

Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

Activating Autophagy by Aerobic Exercise in Mice

自噬活化是在预防许多疾病有益。一种生理方法的诱导自噬体内是体育锻炼。在这里，我们将展示如何通过有氧运动来激活自噬和在小鼠体内测量细胞自噬水平。

小鼠中自噬激活有氧运动

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

activating-autophagy-by-aerobic-exercise-in-mice

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.7K 次观看.  Northwestern University. 自噬活化是在预防许多疾病有益。一种生理方法的诱导自噬体内是体育锻炼。在这里，我们将展示如何通过有氧运动来激活自噬和在小鼠体内测量细胞自噬水平。 

视频: Activating Autophagy by Aerobic Exercise in Mice

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

为控制植物组织分解好氧菌的琼脂块缩影

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

科研

生物学

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

确定的能源系统，在运动的贡献

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

自噬的分析<em&gt;产黄青霉</em&gt;通过结合使用饥饿垫荧光显微镜

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

自噬研究中蛋白质-蛋白质相互作用的研究

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

乳酸脱氢酶螯合测定--一种简便、可靠的测定哺乳动物细胞中自噬性螯合活性的方法

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

微穿刺2光子显微镜辅助小鼠鲍曼空间的研究

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...