Name: assessing autophagic flux by measuring lc3, p62, and lamp1 co-localization using multispectral imaging flow cytometry
Uploaded: 2017-07-21T13:00:00.000+00:00
Duration: 11 min 39 s
Description: Watch this Scientific Journal Video about Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry at JoVE.com

感谢MilliporeSigma的同事Philip J. Morrissey和Sherree L. Friend对他们多年来的支持和指导。我还要感谢Vidya Venkatachalam和Bryan R. Davidson在IDEAS软件中的BDC3功能的帮助， Ryan P. Jessup帮助编辑这本手稿，Raymond Kong在拍摄当天的帮助，特别感谢化妆师辛西娅Xamonthiene。

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我是MilliporeSigma的工​​作，MilliporeSigma是Amnis品牌ImageStream的制造商，在这项研究中使用。

执行此分析时，需要考虑几件事情。重要的是要有适当的对照样品。对于该实验，使用两种不同的对照:对照和对照+氯喹。对照样品用于设定区域的阈值。它代表自噬的基础水平，而不会阻止溶酶体降解，并且是饥饿样品的对照。然而，对照样品不是适用于与Starved + Chloroquine样品相比较的对照，因为氯喹本身会影响对照样品。因此，有必要使用Control + Chloroquine样品。 在对自噬进行任何分析之前，重要的是仅对焦点上的单个单元格（ 即，梯度RMS大于60）进行分析/门控，因为如果存在多个单元格或图像是可能会影响结果失焦了自噬体至关重要es和溶酶体正在关注适当的斑点计数和BDC3分析。在自噬分析之前必须去除凋亡细胞，因为它们可能会使结果偏斜，不应包括在内。对于所有三种标记物（ 即 LC3，p62和LAMP1）都应有适当的染色。例如，所有三个标记都是细胞内的，应该有点状信号;如果信号没有点状，或者如果它在细胞表面，则染色不合适，并且重新实验/染色。另外，对于BDC3来说，对于所有三种感兴趣的荧光标记来说，对于对于明亮的细胞是必要的。需要对积极事件进行选择，以防止BDC3对非特异性结合，成像伪像和/或噪声的测量。这是至关重要的，因为BDC3可以潜在地放大不是真实信号的伪像。由于BDC3只能在亮阳性事件上执行，因此许多单元可能不包括在最终分析中;这是espec对于没有LC3，p62和/或LAMP1的任何积累的对照细胞是真实的。因此，该测定的局限性是BDC3仅检测对于所有三种标记物呈阳性的细胞，这可能不适用于所有自噬实验。 像BDS，先前已经报告了15，16，17，20，21，22，23，24，25，26，BDC3本身并没有考虑到自噬细胞器共定位的数量，从而导致很大程度的自噬数量的变异性。因此，Rajan 等人提出的双变量方法被雇用看一下双变量图，可以问一下对于LC3，p62和LAMP1，细胞必须是阳性的;为什么有这么多的细胞有0个点？这是因为LC3信号可能足够亮以进行共定位，但它可能不符合由峰值掩模（Peak（M11，Ch11-LC3-AF647，Bright，4））设置的阈值，它被认为是点。 本文提出的方案使用MIFC来计数LC3点位点和三个自噬标记物的共定位以测量自噬通量。在基础条件（对照样本）下，自噬体数量少，"高点"发现少数细胞。随着加入抑制自噬体/溶酶体融合的氯喹，LC3斑点数量增加。由于溶酶体不能分解自噬体和位于自噬体中的p62，因此导致LC3，p62和LAMP1自体聚集体积累的共定位增加。这种作用在营养物starvat下扩增离子，诱导自噬。然而，没有添加氯喹，自噬体数量没有显着增加，这很可能是由于自噬周转率的增加。当细胞在氯喹存在下饥饿时，自噬体的共定位和数量有所增加，这支持了饥饿增加自噬通量的结论。已知EBSS是自噬的强大诱导剂。因此，预计增幅会很大。如果使用另一种方法，例如药物诱导来诱导自噬，则对照和处理的样品之间的差异可能更为微妙。 该特定方案被设计用于测量人类细胞系中的自噬，但该测定可以通过切换到该特定物种的抗体来适应其他物种。此外，分析方法可用于需要共定位的任何细胞内应用的三个探针/标记。

Macroautophagy（以下简称自噬）是一种分解代谢途径，其中损伤或功能失调的成分，长寿命蛋白质和细胞器通过溶酶体降解并被回收1 。自噬是一种动态的，多步骤的过程，涉及形成自噬体;自噬体与溶酶体的融合，形成自溶胶体;以及自溶胶体2含量的降解。用于鉴定哺乳动物系统自噬的关键生物学标记是构成自噬体膜的微管相关蛋白1A / 1B-轻链3（LC3）。在自噬过程中，胞质LC3-1与磷脂酰乙醇胺缀合形成LC3-II;然后将LC3-II并入自噬体膜3中 。自噬通量的另一个广泛使用的标记是自噬受体结合体1（SQSTM1，p62）NKS自噬货物到自噬膜4,5。 传统上用于测量自噬的方法是Western印迹和荧光显微术7 。然而，这些方法都没有被认为是"金标准"，2，6为有以两者的这些技术相关联的挑战。因为使用匀浆样品，蛋白质印迹只能给出平均值，所以观察者看不到单个细胞发生了什么。另一方面，荧光显微镜在单细胞水平上给出观察者信息，但缺乏通量能力，难以获得客观和统计学上严格的评估。近年来，通过多光谱成像流式细胞术测定LC3和p62（MIFC，见材料表 ）已经获得了广泛的应用由于其定量功率，高吞吐能力，复用电位以及对每个单元进行成像的能力，因此无法实现。其中最广泛使用的方法使用MIFC，以其伴侣分析软件一起（参见材料的表 ）来测量自噬，是通过LC3斑点或自噬体8，9，10，11，12，13，14的光点计数，15，16，17。然而，自噬体的增加并不一定意味着自噬增加，因为它也可能代表了过程2的封锁。 LC3-II周转量是通过分析细胞存在而测量自噬通量的有用参数没有溶酶体降解抑制剂，如氯喹。氯喹抑制自噬体的融合到溶酶体，由此通过阻止自噬通量溶酶体降解之前，可能会发生18允许自噬体形成，因为自噬的程度的度量的定量。另外，P62主要由自噬降解，并且如果所述自噬体和其内容的溶酶体降解被阻断，P62的累积预期17,19。 自噬是一个多步骤过程，单独测量LC3或p62不能全面了解细胞中发生的情况。最近的出版物强调，有必要检查自溶酶体2，17，20，21的同时形成。 MIFC是唯一能够通过自噬体的共定位成像到溶酶体15以测量自溶酶体的形成- 17，20，21，22，23，24，25，26。此外，还研究了使用MIFC的p62和LC3的共定位以测量自噬通量5 。在参考分析软件中，测量共定位的"特征"被称为"明细相似性R3"（BDS），并且被特别设计为比较两个图像的小而明亮的图像细节。 BDS是半径为3像素或更小的局部亮点的对数转换Pearson相关系数;换句话说，BDS计算o的程度来自两个不同荧光通道的抖动像素。亮点是相关的（ 即相同的空间位置）或不相关的（ 即，不同的空间位置）。因此，相关系数在0（不相关）和1（完全相关）之间变化。的系数是对数转化为增加的范围内的零和无穷大26，27之间。单独的BDS可能不足够; Rajan 等人发现仅使用BDS可能导致假阳性或假阴性结果21 。 BDS评估两种自噬标记的共定位，但不考虑自噬体的数量。为了说明自噬体的数量，Rajan 等包括LC3斑点21的点数计数。 Rajan 等人提出使用LC3点数与BDS的双变量散点图。使用这个双变量的情节，两个人都在e首先确定:一个细胞具有高水平的LC3斑点，一个细胞具有低水平的LC3斑点。高-LC3斑点人口进一步分为两个群体:细胞低共定位（ 即，自噬体的累积）和细胞具有高的共定位（ 即，自体溶酶体的积累）。这种双变量图可以区分具有非常少的自噬体的细胞和具有自噬体和/或自溶质体21的积累的细胞。 到目前为止，使用MIFC伴随分析软件中的单个"特征类型"（参见材料表 ），LC3，p62和LAMP1（溶酶体标记）的同时共定位是不可能的。然而，6.1版中最近推出的一个新功能叫做Bright Detail Colocalization 3（BDC3）。 BDC3比较三个图像中每个图像的明亮细节图像（在本例中为LC）3，p62和LAMP1）并量化三个探针（ 即 LC3，p62和LAMP1）的共定位。 BDC3功能计算修改后的Pearson相关系数以扩展到三个图像。由于三个图像中的亮点是相关或不相关的，相关系数在0（不相关）和1（完全相关）之间变化。系数进行对数转换，以增加0和无穷大之间的动态范围。通过使用BDC3功能切换BDS功能，Rajan 等人提出的分析方法现在可以使用三种最常用的标记来同时测量一个系统中的自噬。在单次测定中将这三种自噬标记共同定位的能力可以导致对自噬的诱导和调节的新见解。以下方案概述了诱导Jurkat细胞自噬的步骤。用LC3，p62和LAMP1抗体标记细胞;获取有关多数据的数据口腔成像流式细胞仪;并分析数据以评估自噬通量。

<table><tbody><tr><td>15 mL centrifuge tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>AF647 labeled Donkey anti-Mouse - IgG&amp;nbsp;</td><td>Invitrogen</td><td>A-31571</td><td>Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647. Concntration 2 mg/mL. https://www.thermofisher.com/antibody/product/Donkey-anti-Mouse-IgG-H-L-Secondary-Antibody-Polyclonal/A-31571</td></tr><tr><td>anti-human CD107a-PE (LAMP1)</td><td>BioLegend</td><td>328607</td><td>Clone H4A3. Concentration 400 &amp;micro;g/mL. http://www.biolegend.com/pe-anti-human-cd107a-lamp-1-antibody-4967.html</td></tr><tr><td>anti-human CD45- AF488</td><td>BioLegend</td><td>304017</td><td>Clone HI30. http://www.biolegend.com/alexa-fluor-488-anti-human-cd45-antibody-2738.html</td></tr><tr><td>anti-human CD45- PE&amp;nbsp;</td><td>BioLegend</td><td>304008</td><td>Clone HI30.&amp;nbsp; http://www.biolegend.com/pe-anti-human-cd45-antibody-708.html</td></tr><tr><td>anti-human CD45-AF647&amp;nbsp;</td><td>BioLegend</td><td>304018</td><td>Clone HI30.&amp;nbsp; http://www.biolegend.com/alexa-fluor-647-anti-human-cd45-antibody-2739.html</td></tr><tr><td>Anti-LC3 (Human) mAb clone 4E12</td><td>MBL International Corporation</td><td>M152-3</td><td>Clone 4E+12.&amp;nbsp; Concentration 2 mg/mL.&amp;nbsp; https://www.mblintl.com/products/m152-3</td></tr><tr><td>Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (Alexa Fluor 488)</td><td>abcam</td><td>ab185015</td><td>Clone EPR4844.&amp;nbsp; Concentration 0.5 mg/mL.&amp;nbsp; http://www.abcam.com/sqstm1-p62-antibody-epr4844-autophagosome-marker-alexa-fluor-488-ab185015.html</td></tr><tr><td>Chloroquine diphosphate Salt</td><td>Sigma-Aldrich</td><td>C6628-25G</td><td></td></tr><tr><td>Cleanser - Coulter Clenz&amp;nbsp;</td><td>Beckman Coulter</td><td>8546931</td><td>Fill container with 200 mL of Cleanser.&amp;nbsp; https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=8546931#2/10//0/25/1/0/asc/2/8546931///0/1//0/</td></tr><tr><td>DAPI Stain 5 mg/mL</td><td>MilliporeSigma</td><td>508741</td><td>http://www.emdmillipore.com/US/en/product/DAPI-Stain---CAS-28718-90-3---Calbiochem,EMD_BIO-508741</td></tr><tr><td>Debubbler - 70% Isopropanol</td><td>MilliporeSigma</td><td>1.3704</td><td>Fill container with 200 mL of Debubbler.&amp;nbsp; http://www.emdmillipore.com/US/en/product/2-Propanol-70%25-%28V%2FV%29-0.1-%C2%B5m-filtred,MDA_CHEM-137040?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline 10x</td><td>MilliporeSigma</td><td>BSS-2010-B</td><td>&amp;nbsp;Ca++Mg++ Free&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline 1x</td><td>MilliporeSigma</td><td>BSS-1006-B</td><td>PBS Ca++Mg++ Free&amp;nbsp;</td></tr><tr><td>Earle&#039;s Balanced Salt Solution 1x</td><td>Gibco</td><td>14155</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>HyClone</td><td>SH30071.03</td><td></td></tr><tr><td>Formaldehyde, 10%, methanol free, Ultra Pure</td><td>Polysciences, Inc.</td><td>04018</td><td>This is what is used for the 4% and 1% Formalin. CAUTION: Formalin/Formaldehyde toxic by inhalation and if swallowed.&amp;nbsp; Irritating to the eyes, respiratory systems and skin.&amp;nbsp; May cause sensitization by inhalation or skin contact. Risk of serious damage to eyes.&amp;nbsp; Potential cancer hazard.&amp;nbsp; http://www.polysciences.com/default/catalog-products/life-sciences/histology-microscopy/fixatives/formaldehydes/formaldehyde-10-methanol-free-pure/</td></tr><tr><td>Hanks&#039; Balanced Salt Solution 1x</td><td>Gibco</td><td>14175</td><td></td></tr><tr><td>Jurkat, Clone E6-1 (ATCC TIB-152)</td><td>ATCC</td><td>TIB-152</td><td>https://www.atcc.org/products/all/TIB-152.aspx</td></tr><tr><td>MEM Non-Essential Amino Acids 100x</td><td>HyClone</td><td>SH30238.01</td><td></td></tr><tr><td>MIFC - ImageStreamX Mark II</td><td>MilliporeSigma</td><td>100220</td><td>A 2 camera ImageStreamX Mark II eqiped with the 405nm, 488nm, and 642nm lasers was used. http://www.emdmillipore.com/US/en/life-science-research/cell-analysis/amnis-imaging-flow-cytometers/imagestreamx-Mark-ii-imaging-flow-cytometer/VaSb.qB.QokAAAFLzRop.zHe,nav?cid=BI-XX-BDS-P-GOOG-FLOW-B325-0006</td></tr><tr><td>MIFC analysis software - IDEAS 6.2</td><td>MilliporeSigma</td><td>100220</td><td>The companion software to the MIFC (ImageStreamX MKII).&amp;nbsp; IDEAS version 6.2</td></tr><tr><td>MIFC software - INSPIRE</td><td>MilliporeSigma</td><td>100220</td><td>This is the software that runs the MIFC (ImageStreamX MKII). INSPIRE version 200.1.388.0</td></tr><tr><td>PE anti-human CD107a (LAMP-1) Antibody clone H4A3</td><td>BioLegend</td><td>328608</td><td>http://www.biolegend.com/pe-anti-human-cd107a-lamp-1-antibody-4967.html</td></tr><tr><td>Penicllin/Streptomycin/Glutamine solution 100x</td><td>HyClone</td><td>SV30082.1</td><td></td></tr><tr><td>Rinse - Ultrapure water or deionized water</td><td>NA</td><td>NA</td><td>You can use any ultrapure water or deionized water.&amp;nbsp; Fill container with 900 mL of Rinse.</td></tr><tr><td>RPMI-1640 Medium 1x</td><td>HyClone</td><td>SH30027.01</td><td></td></tr><tr><td>Sheath - PBS</td><td>MilliporeSigma</td><td>BSS-1006-B</td><td>This is the same as Dulbecco&#039;s Phosphate Buffered Saline 1X&amp;nbsp; Ca++MG++ free.&amp;nbsp; Fill container with 900 mL of Sheath.</td></tr><tr><td>Siliconized polypropylene microcentrifuge tubes</td><td>Fisherbrand</td><td>02-681-320</td><td>Fisherbran Siliconized Low-Retention Microcentrifuge Tubes 1.5 mL. https://www.fishersci.com/shop/products/fisherbrand-siliconized-low-retention-microcentrifuge-tubes-8/p-193936</td></tr><tr><td>Sodium Pyruvate solution (100 mM)</td><td>HyClone</td><td>SH30239.01</td><td></td></tr><tr><td>Sterilizer - 0.4 - 0.7% Hypochlorite</td><td>VWR</td><td>JT9416-1</td><td>This is assentually 10% Clorox bleach that can be made by deluting Clorox bleach with water.&amp;nbsp; Fill container with 200 mL of Sterilzer.</td></tr><tr><td>System Calibration Reagent - SpeedBead</td><td>MilliporeSigma</td><td>400041</td><td>Each tube holds ~10 mL.&amp;nbsp; https://www.emdmillipore.com/US/en/life-science-research/cell-analysis/amnis-imaging-flow-cytometers/support-training/XDqb.qB.wQMAAAFLBDUp.zHu,nav&amp;nbsp;</td></tr><tr><td>T75 flask</td><td>Falcon</td><td>353136</td><td></td></tr><tr><td>TRITON X-100, PROTEIN GRADE Detergent, 10% Solution, Sterile-Filtered</td><td>MilliporeSigma</td><td>648463-50ML</td><td>http://www.emdmillipore.com/US/en/product/TRITON-X-100,-PROTEIN-GRADE-Detergent,-10%25-Solution,-Sterile-Filtered---CAS-9002-93-1---Calbiochem,EMD_BIO-648463</td></tr><tr><td>Water, Cell Culture Grade&amp;nbsp;</td><td>HyClone</td><td>SH30529.03</td><td></td></tr></tbody></table>

assessing autophagic flux by measuring lc3, p62, and lamp1 co-localization using multispectral imaging flow cytometry

自噬是分解代谢途径，其中在生长和分化期间积累的正常或功能失调的细胞组分通过溶酶体降解并被再循环。在自噬过程中，细胞质LC3蛋白被脂质化并引入自噬体膜。然后自噬体与溶酶体融合以形成自体溶质，其中发生自噬体囊泡及其内含物的分解。与LC3结合的泛素相关蛋白p62也用于监测自噬通量。进行自噬的细胞应该证明p62，LC3和溶酶体标记物的共定位。已经使用免疫荧光显微镜来以每细胞为基础目视鉴定LC3斑点，p62和/或溶酶体。然而，客观和统计上严格的评估可能难以获得。为了克服这些问题，使用多光谱成像流式细胞术以及比较光亮度的分析特征来自三个自噬标记（LC3，p62和溶酶体LAMP1）的尾部图像，并量化它们的共定位，结合LC3点计数，以客观，定量和统计学鲁棒的方式测量自噬。

该分析方法使用多个特征来评估自噬通量。为了充分了解最终的双变量图，必须首先对个别分析特征进行调查。自噬的计数是衡量自噬的一种合乎逻辑的方法;然而，LC3斑点的大小/形状/亮度可以在细胞之间显着变化。变异可能使得在分析软件中手动计数自噬体或使用点数特征难以计数。因此，由于自噬体的大变异性，无斑点特征将是完美的。然而，大多数单元格26都能实现良好的点数功能。 图3示出了使用不同的斑点掩模的Jurkat细胞中LC3斑点的斑点掩蔽的实例。为此数据集选择的点计数功能是Spot Count_Peak（M11，Ch11-LC3-AF647，Bright，4）_4，这意味着点计数功能计数为pe在通道11（Ch11-LC3-AF647）上的默认通道11掩码（M11）中标识的ak掩码，点到单元背景比为4（Bright，4）。 图4显示了用反LC3-AF647标记的对照，对照+氯喹，饥饿和饥饿+氯喹Jurkat细胞的斑点计数直方图和代表性图像。控制和饥饿平均斑点数没有显着差异;与氯喹相比，对照+氯喹与饥饿+氯喹相比，平均值差异很大。 下一个调查功能是BDC3。 BDC3是三种标记物/探针的共定位的测量，在这种情况下，LC3，p62和LAMP1。 图5A - 5D显示了用抗p62-AF488标记的对照，对照+氯喹，饥饿和饥饿+氯喹Jurkat细胞的BDC3直方图，抗LAMP1-PE和抗LC3-AF647。控制平均值与饥饿平均值之间存在转移，以及对照+氯喹平均值与饥饿+氯喹平均值之间的偏差。然而，从图5E - 5H中的平均BDC3评分中观察细胞的图像，样本之间的差异比直方图可能导致人们相信更大。这是因为BDC3不考虑自噬细胞器的数量，导致相同BDC3评分的自噬体数量的变化程度很大。在大多数情况下，所有三个探针之间存在重叠，因为即使在基础水平，p62，LAMP1和LC3应在一定程度上共同定位或驻留在细胞中的相似区域。相比之下，不能共定位的三个探针的实例是用于Starved + Chloroquine样品的抗p62-AF488，抗LC3-AF647和DAPI核染料， 如图6所示。/ P&gt; 当点计数特征和BDC3特征相结合时，存在提高区别各种样本/条件的能力的不同亚群的存在是明显的。 图7显示了四个样品的LC3与BDC3 p62 / LAMP1 / LC3的斑点数的双变量图:对照，对照+氯喹，饥饿和饥饿+氯喹。对照样品用于设置三个群体的门控策略:低斑点，高斑点/低BDC3和高斑点/高BDC3。对照样品显示大于98％的细胞具有1个或更少的斑点。高点/低BDC3和高点/高BDC3之间的边界被设置为BDC3得分为1，因为对照样本的91％以上的BDC3得分小于1.双变量图的结果总结如表1所示。 年龄= "1"&gt; <img alt="图1" class="xfigimg" src="/files/ftp_upload/55637/55637fig1.jpg" /> 图1:MIFC仪器设置。本实验步骤8中概述的MIFC仪器设置的屏幕截图。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55637/55637fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55637/55637fig2.jpg" /> 图2:分析软件门控策略。分析软件选通方案的屏幕截图，在协议的步骤9中概述。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55637/55637fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> fig3.jpg"/&gt; 图3:LC3斑点掩蔽。 Jurkat细胞图像和掩模用于创建点计数功能。显示为BrightField（BF），LC3-AF647，Peak（M11，Ch11-LC3-AF647，Bright，2），Peak（M11，Ch11-LC3-AF647，Bright，4），Peak（M11，Ch11-LC3-AF647 ，Bright，5），Spot（M11，Ch11-LC3-AF647，Bright，5,3,1）和Spot（M11，Ch11-LC3-AF647，Bright，6,2,1）。最适合所有细胞的面膜为Peak（M11，Ch11-LC3-AF647，Bright，4）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55637/55637fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55637/55637fig4.jpg" /> 图4:LC3点数直方图。使用点计数功能Spot Count_Peak（M11，Ch11-LC3-AF647，Bright，4）_4和Control（ A ，浅蓝色），Control + Chloroquin的LC3-AF647点计数直方图e（ B ，蓝色），饥饿（ C ，粉红色）和饥饿+氯喹（ D ，红色）。对照，对照+氯喹，饥饿和饥饿+氯喹的平均斑数分别为0.07,1.13,10.10和2.77。 BF，LC3-AF647（红色），DAPI核染料（蓝色）和代表性细胞的LC3-AF647和DAPI图像的平均斑点数的复合物显示为对照（ E ，0个斑点），对照+氯喹F ，1点），饥饿（ G ，0个点）和饥饿+氯喹（ H ，3个点）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55637/55637fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55637/55637fig5.jpg" /> 图5:p62 / LAMP1 / LC3的BDC3直方图。 BDC3 p62 / LAMP1 / LC3直方图对照（ A ，淡蓝色），对照+氯喹（ B ，蓝色），饥饿（ C ，粉红色）和饥饿+氯喹（ D ，红色）。对照，对照+氯喹，饥饿和饥饿+氯喹的平均BDC3评分分别为0.57,0.82,0.74和0.98。 BF; p62-AF488（绿色）; LAMP1-PE（黄色）; LC3-AF647（红色）;对照（ E ），对照+氯喹（ F ），饥饿（ G ），饥饿+氯喹（ F ），饥饿+氯喹H ）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55637/55637fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/55637/55637fig6.jpg" /> 图6:p62 / L的BDC3直方图C3 / DAPI为饥饿+氯喹样品。 （ A ）显示了Starved + Chloroquine样品的BDC3 p62 / LC3 / DAPI直方图。平均BDC3评分为0.07。 （ B ）BF; p62-AF488（绿色）; LC3-AF647（红色）; DAPI（蓝色）;对于Starved + Chloroquine样品，显示了平均BDC3的代表性细胞的p62-AF488，LC3-AF647和DAPI图像的复合物。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55637/55637fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图7" class="xfigimg" src="/files/ftp_upload/55637/55637fig7.jpg" /> 图7:LC3点数与BDC3 p62 / LAMP1 / LC3的双变量图。 （ A ）对照，对照+氯喹，饥饿和饥饿+氯喹Jurkat细胞的LC3斑点数与BDC3 p62 / LAMP1 / LC3的双变量图。 （ B ）BF; p62-AF488（绿色）; LAMP1-PE（黄色）; LC3-AF647（红色）;对于Starved + Chloroquine样品，显示了来自三个区域（ 即，低斑点，高斑点/低BDC3和高斑点/高BDC3）的p62-AF488，LAMP1-PE和LC3-AF647图像的复合物。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55637/55637fig7large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td>细胞计数</td><td>亮度细节共定位3平均值</td><td>点数LC3平均值</td><td> ％低点</td><td> ％高点/低BDC3 </td><td> ％高点/高BDC3 </td></tr><tr><td>控制</td><td> 439 </td><td> 0.57 </td><td> 0.07 </td><td> 98.2 </td><td> 1.8 </td><td> 0.0 </td></tr><tr><td>对照+氯喹</td><td> 1432 </td><td> 0.82 </td><td> 1.13 </td><td> 68.9 </TD&gt; <td> 19.5 </td><td> 11.7 </td></tr><tr><td>饥饿</td><td> 1204 </td><td> 0.74 </td><td> 0.10 </td><td> 98.4 </td><td> 0.6 </td><td> 1.0 </td></tr><tr><td>饥饿+氯喹</td><td> 1811 </td><td> 0.98 </td><td> 2.77 </td><td> 32.1 </td><td> 36.9 </td><td> 31.0 </td></tr></tbody></table> 表1:Jurkat LC3点数与 BDC3 p62 / LAMP1 / LC3双变量图的总结

Watch this Scientific Journal Video about Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry at JoVE.com

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

在这里，具有分析特征的多光谱成像流式细胞术可以比较3个自噬标记的明亮细节图像，并量化其共定位以及LC3斑点计数，用于以客观，定量和统计学鲁棒的方式测量自噬。

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

assessing-autophagic-flux-measuring-lc3-p62-lamp1-co-localization

Universidad San Sebastian

Research

JoVE Journal

Biology

31.1K 次观看.  MilliporeSigma. 在这里，具有分析特征的多光谱成像流式细胞术可以比较3个自噬标记的明亮细节图像，并量化其共定位以及LC3斑点计数，用于以客观，定量和统计学鲁棒的方式测量自噬。 

视频: Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

科研

生物学

<meta charset="utf8"></head><body>评估溶酶体融合受损自噬中 LC3-II 阳性 EV 释放

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited.
To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.

<meta charset="utf8"></head><body>通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

Here, we present the methodology for concisely assessing autophagosome marker LC3-II levels in extracellular vesicles (EVs) by immunoblotting. Analysis for LC3-II levels in EVs, autolysosome formation, and omegasome formation suggests the new role of STX6 in the release of LC3-II-positive EVs when autophagosome-lysosome fusion is inhibited.

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf ...

生物化学

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

医学

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

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 ...

Measuring Diurnal Rhythms in Autophagic and Proteasomal Flux

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main lysosome-dependent pathway is called autophagy, while the primary non-lysosomal method for protein catabolism is the ubiquitin-proteasome system. Recent studies in model organisms suggest that the activity of both autophagy and the ubiquitin-proteasome system is not constant across the day but instead varies according to a daily (circadian) rhythm. The ability to measure biological rhythms in protein turnover is important for understanding how cellular quality control is achieved and for understanding the dynamics of specific proteins of interest. Here we present a standardized protocol for quantifying autophagic and proteasomal flux in vivo that captures the circadian component of protein turnover. Our protocol includes details for mouse handling, tissue processing, fractionation, and autophagic flux quantification using mouse liver as the starting material.

We describe our protocol for measuring biological rhythms in protein catabolism via autophagy and the proteasome in mouse liver.

测量自噬和前列腺通量中的日节律

Cells employ several methods for recycling unwanted proteins and other material, including lysosomal and non-lysosomal pathways. The main ...

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

This paper details monitoring autophagy flux to identify new molecules by cell-based imaging screening.

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify ...

通过使用多光谱成像流式细胞术测量LC3，p62和LAMP1共定位评估自噬通量

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通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

通过细胞外囊泡释放 LC3-II 与细胞内 LC3 阳性囊泡积累的关系的评价

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Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis