Hochecker Barbara, Matt Katja C, Meßmer Alica L, Scherer Melanie M, Bergemann Jörg
Department of Life Sciences, Albstadt-Sigmaringen University of Applied Sciences, Sigmaringen, Germany.
Bio Protoc. 2024 Jul 5;14(13):e5025. doi: 10.21769/BioProtoc.5025.
autophagosomal compartments is required that allows comparison of autophagosomal activity under different conditions. Existing methods such as western blotting and immunohistochemistry for analysing the location and relative abundance of intracellular proteins associated with autophagy, or transmission electron microscopy (TEM), which are either very time-consuming, expensive, or both, are less suitable for this purpose. The method described in this protocol allows the absolute quantification of autophagosomes per cell in human fibroblasts using the CYTO-ID® Autophagy Detection Kit after heat therapy compared to a control. The Cyto-ID® assay is based on the use of a specific dye that selectively stains autophagic compartments, combined with an additional Hoechst 33342 dye for nuclear staining. The subsequent recognition of these stained compartments by the Cytation Imager enables the software to determine the number of autophagosomes per nucleus in living cells. Additionally, this absolute quantification uses an image-based method, and the protocol is easy to use and not time-consuming. Furthermore, the method is not only suitable for heat therapy but can also be adapted to any other desired therapy or substance. Key features • Absolute quantification of autophagic compartments in living cells. • Optimised protocol for the determination of autophagy in primary human skin fibroblasts. • Allows the testing of active substances and treatments concerning autophagy. • Imaging-based method for the determination of autophagy.
需要有自噬体区室，以便能够比较不同条件下的自噬体活性。现有的方法，如用于分析与自噬相关的细胞内蛋白质的定位和相对丰度的蛋白质免疫印迹法和免疫组织化学法，或者透射电子显微镜（TEM），要么非常耗时，要么成本高昂，或者两者兼具，不太适合此目的。本方案中描述的方法允许在热疗后，使用CYTO-ID®自噬检测试剂盒对人成纤维细胞中每个细胞的自噬体进行绝对定量，并与对照进行比较。CYTO-ID®检测基于使用一种特异性染料，该染料可选择性地对自噬区室进行染色，并结合另外一种用于细胞核染色的Hoechst 33342染料。随后，Cytation成像仪对这些染色区室的识别使软件能够确定活细胞中每个细胞核的自噬体数量。此外，这种绝对定量使用基于图像的方法，该方案易于使用且不耗时。此外，该方法不仅适用于热疗，还可适用于任何其他所需的治疗或物质。关键特性 • 活细胞中自噬区室的绝对定量。 • 用于测定原代人皮肤成纤维细胞自噬的优化方案。 • 允许测试与自噬有关的活性物质和治疗方法。 • 基于成像的自噬测定方法。
