Name: evaluating autophagy levels in two different pancreatic cell models using lc3 immunofluorescence
Uploaded: 2023-04-28T13:40:00
Description: Watch this Scientific Journal Video about Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence at JoVE.com

这项工作得到了布宜诺斯艾利斯大学（UBACyT 2018-2020 20020170100082BA）、国家科学研究与技术委员会（CONICET）（PIP 2021-2023 GI− 11220200101549CO;和PUE 22920170100033）和国家科学技术促进局（PICT 2019-01664）的资助。

1. 细胞制备
<ol><li>将12毫米圆形盖玻片浸泡在无水乙醇中，并将它们垂直放置在24孔板的孔中。</li>
	<li>取下盖子，将多孔板暴露在紫外线辐射下15分钟。</li>
	<li>水平放置盖玻片，并用Dulbecco的改良鹰培养基（DMEM）清洗它们。</li>
	<li>播种低传代数的胰腺细胞。应调整量，以在固定当天获得50%-75%的汇合度16。 注意：建议每孔接种2.5×104 PANC-1或4个×104 AR4...

<ol>
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该协议中描述的方法允许可视化细胞中的内源性LC3分布并量化不同条件下的自噬水平。另一种用于分析LC3分布和确定自噬激活的类似方法涉及荧光标记的LC3转染（如RFP-LC3）19。RFP-LC3转染的优点是不需要固定（允许将该方法应用于活细胞成像20），更便宜，并且不依赖于LC3抗体反应性。另一方面，LC3的免疫荧光具有提供内源性LC3图像的优点，从而避免了与LC3过表?...

巨自噬（通常称为自噬）是一种特殊的分解代谢过程，可选择性降解细胞质成分，包括蛋白质和受损细胞器1，2。自噬允许细胞对应激刺激做出生理反应，从而维持细胞稳态3。在自噬过程中，形成双膜囊泡：自噬体。自噬体包含货物分子并驱动它们到溶酶体进行降解1，4。 
自噬体由自噬蛋白微管相关蛋白轻链3（LC3）5修饰。当不诱导自噬时，LC3以LC3-I构象扩散在细胞质和细胞核中。另一方面，当诱导自噬时，LC3与自噬结构6中的磷脂酰乙醇胺偶联。这种新的LC3构象被称为LC3-II1。LC3构象转移导致其细胞定位及其十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）迁移发生变化，这可以通过免疫荧光和蛋白质印迹5，7等技术检测到。通过这种方式，LC3偶联是自噬过程中的关键事件，可用于测量自噬活性。
胰腺腺泡细胞是一种高度分化的细胞，在健康条件下，自噬率较低。然而，在不同的生理条件下或在药物刺激下，它们可以激活自噬。因此，测定该细胞系中的自噬水平有助于研究不同药理学或生物制剂对自噬的潜在直接或间接影响8，9。
导管胰腺腺癌是最致命的癌症类型之一，因为它诊断较晚，化疗耐药性高10。由于自噬相关蛋白的转录上调和翻译后活化，胰腺癌细胞具有高自噬活性11。胰腺癌细胞可能会调整其自噬水平，以应对缺氧、营养剥夺或化疗引起的损伤等不利条件11。因此，分析胰腺癌细胞中的自噬水平可以帮助了解它们如何适应不同的环境，并评估自噬调节治疗的有效性。
这项研究展示了一种在两种不同的胰腺细胞模型中进行LC3免疫荧光的方法。第一个模型PANC-1细胞作为胰腺导管腺癌的模型。这些细胞用吉西他滨处理，吉西他滨是一种化疗药物，以前已被证明可以诱导自噬，特别是在携带致癌Kirsten大鼠肉瘤病毒基因（KRAS）的胰腺癌细胞中12，13。第二种模型AR42J细胞作为胰腺外分泌细胞的更生理模型。这些细胞用地塞米松分化，使其与腺泡胰腺细胞更相似14。在这些细胞中，通过使用PP242（一种有效的mTOR抑制剂15）在药理学上诱导自噬。在这项研究中，我们证明了用两种不同的胰腺模型描述的方案的适用性及其区分低自噬和高自噬状态的能力。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Phosphate-Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm round coverslips</td>
			<td>HDA</td>
			<td>CBR_OBJ_6467</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Well- Cell Culture Plate</td>
			<td>Sorfa</td>
			<td>220300</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Biopack</td>
			<td>2207.10.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>R37119</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEN</td>
			<td>Sartorius</td>
			<td>01-052-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>NATOCOR&#160;</td>
			<td>Lintc-634</td>
			<td></td>
		</tr>
		<tr>
			<td>Gemcitabina</td>
			<td>Eli Lilly</td>
			<td>VL7502</td>
			<td></td>
		</tr>
		<tr>
			<td>LC3B (D11) XP Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>3868S</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Anedra</td>
			<td>6197</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm &#34;M&#34; (Laboratory Sealing Film)</td>
			<td>Bemis/Curwood</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep Solution</td>
			<td>Sartorius</td>
			<td>03-031-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>PP242</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-301606</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Gibco</td>
			<td>11570626</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating autophagy levels in two different pancreatic cell models using lc3 immunofluorescence

自噬是一种特殊的分解代谢过程，可选择性降解细胞质成分，包括蛋白质和受损细胞器。自噬允许细胞对压力刺激做出生理反应，从而维持细胞稳态。癌细胞可能会调节其自噬水平以适应不良条件，如缺氧、营养缺乏或化疗造成的损害。导管胰腺腺癌是最致命的癌症类型之一。胰腺癌细胞由于自噬蛋白的转录上调和翻译后激活而具有高自噬活性。
这里，PANC-1细胞系被用作胰腺人癌细胞的模型，AR42J胰腺腺泡细胞系被用作高度分化的哺乳动物细胞的生理模型。本研究使用微管相关蛋白轻链3（LC3）的免疫荧光作为自噬激活状态的指标。LC3是一种自噬蛋白，在基础条件下，在细胞质中显示出弥漫分布模式（在这种情况下称为LC3-I）。自噬诱导触发LC3与新形成的自噬体表面的磷脂酰乙醇胺的结合，形成LC3-II，这是一种有助于自噬体形成和扩增的膜结合蛋白。为了量化标记的自噬结构的数量，在“3D对象计数器”工具的帮助下使用了开源软件FIJI。
生理条件和癌细胞中自噬水平的测量使我们能够研究在缺氧、化疗或敲低某些蛋白质等不同条件下自噬的调节。

该方案在胰腺细胞系中对LC3进行免疫荧光，以确定不同条件下的自噬水平。该实验的结果是从对应于LC3和DAPI的红色和蓝色通道获得细胞图像。LC3图像显示该蛋白的细胞分布，而DAPI显示核定位。 图2A 显示了在基础或吉西他滨处理条件下LC3的免疫荧光及其与PANC-1细胞中DAPI染色合并的代表性图像。使用斐济的 3D对象计数器 工具分析了一组LC3染色图像。使用该软件，...

Watch this Scientific Journal Video about Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence at JoVE.com

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence

该协议的目标是通过LC3免疫荧光和LC3点定量确定胰腺癌和胰腺腺泡细胞中的自噬水平。

使用LC3免疫荧光评估两种不同胰腺细胞模型中的自噬水平

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows ...

LC3免疫荧光能够测定胰腺细胞中的自噬水平。它允许研究不同药物对胰腺细胞自噬的潜在影响。免疫荧光是一种用于检测内源性LC3蛋白的技术，可避免过度表达LC3引起的问题，例如形成与自噬无关的蛋白质聚集体。演示该程序的将是博士毕业生Felipe Renna和Maria Ines Vaccaro实验室的博士生Malena Herrera Lopez。要开始细胞制备，将12毫米圆形盖玻片浸泡在无水乙醇中。取下盖子后，将浸泡过的盖玻片垂直放入 24 孔板中。将多孔板暴露在紫外线辐射下 15 分钟。然后将盖玻片水平放置并用DMEM清洗。现在看到胰腺细胞的低传代数，重新悬浮在DMEM中，含有10%FBS，青霉素和链霉素，并将细胞孵育两天。细胞接种两天后，在DMEM中以每微升浓度一微克制备吉西他滨溶液。然后在单独的管中从每个孔中吸出一半体积，并加入适量的吉西他滨溶液。将对应于每个孔的一半体积放回处理过的孔中，并将细胞孵育24小时。为了固定和透化细胞，将冷甲醇加入含有细胞的24孔板中。还要准备一个带有冷PBS的六孔板。使用注射器针头和镊子，取下每张盖玻片并在PBS中清洗两次。然后在甲醇中孵育六分钟。用PBS洗涤两次后，将盖玻片在封闭溶液中孵育一小时。在封闭溶液中以1：1000的比例加入抗LC3，并保持在冰上。在多孔盖上放置一块实验室密封膜，并在密封膜上每张盖玻片上加入 25 微升抗 LC3 溶液。使用注射器针头和镊子，将每个盖玻片放在一抗液滴上，确保细胞侧与溶液接触。将多孔板放入湿度室中。用箔纸盖住，在冰箱中孵育过夜。第二天，从湿度室中取出多孔板。将盖玻片放回多孔板上，并用PBS洗涤三次。在封闭溶液中以1：800的比例稀释荧光标记的抗兔，并在黑暗中保持冰上。密封多孔盖后，在密封膜上每张盖玻片上加入 25 微升抗兔溶液。并如前所述，用二抗处理盖玻片。然后将板在室温下在湿度室中孵育两个小时，避光。在用抗体孵育结束时，用PBS洗涤多孔板上的盖玻片三次。将每个盖玻片与准备好的DAPI溶液孵育10分钟。用PBS洗涤后，将多孔板保持在黑暗中。对于细胞蒙太奇，准备两个装有水和一张纸的烧杯。在载玻片上的每张盖玻片上加入 10 微升 PVA-DABCO 溶液。用水清洗盖玻片，然后在纸上晾干。最后，将其放在PVA-DABCO下降上。并在黑暗中干燥过夜。第二天，使用倒置共聚焦显微镜，可视化标记的细胞并捕获图像。捕获图像后，将包含捕获通道的每个图像文件拖放到斐济屏幕中。在对话框中，单击“确定”以打开图像。然后关闭打开的控制台窗口。从“图像”选项卡中，选择“颜色”，然后选择“拆分通道”。关闭与LC3图像以外的通道对应的图像。然后在“图像”选项卡中，选择“调整”，然后选择“色彩平衡”。将最大滑块向左移动，直到图像饱和以可视化单元格轮廓。使用手绘选择工具绘制单元格轮廓，然后单击重置以调整颜色。要从“编辑”选项卡中剪切所选项目，请选择“剪切”，然后关闭图像而不保存它。然后从编辑选项卡中，选择粘贴。在分析菜单中，选择工具 3D 对象计数器。将阈值设置为 2000，将大小筛选器设置为 50 到 500。确保标记对象和摘要框，然后单击日志窗口 OK.In，点数将被描述为检测到的对象。免疫荧光指示LC3蛋白的细胞分布，而DAPI显示胰腺细胞中的核定位。吉西他滨处理下LC3点显著增加，表明自噬活性。在过度汇合的情况下，外分泌胰腺细胞往往会堆积并相互叠加生长。多聚甲醛细胞固定方法对保存LC3蛋白无效，因为它不能反映准确的分布。当接种后无需等待两天即可进行固定或处理时，PANC1细胞呈圆形。这种形态降低了细胞质和细胞核之间的关系，使得难以理解LC3的细胞内分布。不完全甲醇固定可能会干扰LC3免疫标记的正确性，导致图像不清晰和定量欠佳。在此过程中要记住的最重要的事情是，细胞必须在冷甲醇中固定六分钟，而不是多聚甲醛。按照此过程，可以分析LC3与其他蛋白质之间的共定位，从而可以研究与LC3功能相关的不同分子途径。

Research

JoVE Journal

Biology

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

科研

生物学

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Mouse Sperm Cryopreservation and Recovery using the IÃƒâ€šÃ‚Â·Cryo Kit

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Measurement of Heme Synthesis Levels in Mammalian Cells

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

的三种不同方法对乳腺癌细胞株确定细胞增殖的比较

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Activating Autophagy by Aerobic Exercise in Mice

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 ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

使用 体内 抑制、免疫荧光和流式细胞术对精子细胞中驱动蛋白-7 CENP-E 进行功能评估

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

分析黑 腹果蝇 幼虫脂肪体中饥饿诱导的自噬

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy

内源性LC3的超微结构定位-截面相关光电子显微镜

The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal ...