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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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="http://ecsource.jove.com/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 de Cartagena UDEC

Research

JoVE Journal

Biology

31.0K 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

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

科研

生物工程

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture &#8211; information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

微流控成像流式细胞仪通过不对称检测时间延伸光学显微镜（ATOM）

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

工程学

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

癌症研究

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

免疫与感染

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

一个小鼠红细胞亚群富集去核活动由多光谱成像流式细胞仪鉴定

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

生物学

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

pH 依赖性荧光 Keima 对流式细胞术 Mitophagy 的敏感性测定

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

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 ...