Scherer Melanie, Hochecker Barbara, Matt Katja, Meßmer Alica, Bergemann Jörg
Department of Life Sciences, Albstadt-Sigmaringen University of Applied Sciences, Sigmaringen, Germany.
Bio Protoc. 2025 Sep 20;15(18):e5457. doi: 10.21769/BioProtoc.5457.
Autophagy plays a crucial role in cellular homeostasis and is responsible for removing and degrading damaged cytoplasmic cargo. This lysosome-mediated catabolic process removes defective organelles and misfolded proteins, and impaired autophagy has been directly linked to ageing and numerous diseases. This emphasises the importance of developing intervention methods to counteract this dysregulation. One promising intervention is thermal therapy, specifically hyperthermia, which is described in this protocol. In order to investigate this form of treatment, a rapid and reliable detection method is required to allow comparison of autophagy status under different conditions. While methods such as transmission electron microscopy (TEM) or western blotting can provide valuable structural analysis, they are often time-consuming and expensive, and are not suitable for small, round cells such as peripheral blood mononuclear cells (PBMCs). The method described in this protocol enables absolute quantification of PBMCs using the Guava Autophagy Detection kit after heat treatment with water-filtered infrared-A radiation (wIRA), compared with an untreated control. This method is based on antibody labelling, and subsequent flow cytometric analysis enables the number of autophagosomes to be determined by measuring the FITC intensity. This protocol provides rapid, reliable results and can be adapted to investigate not only heat therapy, but also other interventions, such as caloric restriction. Key features • Rapid and reliable ex vivo quantification of autophagy in living cells. • Optimised protocol for the determination of autophagy in primary human blood cells. • Allows the testing of active substances and treatments concerning autophagy. • Flow cytometry-based method for the determination of autophagy.
自噬在细胞内稳态中起着关键作用，负责清除和降解受损的细胞质物质。这种由溶酶体介导的分解代谢过程可清除有缺陷的细胞器和错误折叠的蛋白质，而自噬功能受损与衰老和多种疾病直接相关。这凸显了开发干预方法以对抗这种失调的重要性。一种有前景的干预措施是热疗法，特别是热疗，本方案对此进行了描述。为了研究这种治疗形式，需要一种快速可靠的检测方法，以便比较不同条件下的自噬状态。虽然诸如透射电子显微镜（TEM）或蛋白质免疫印迹等方法可以提供有价值的结构分析，但它们通常耗时且昂贵，不适用于外周血单核细胞（PBMC）等小而圆的细胞。本方案中描述的方法能够在用水过滤红外-A辐射（wIRA）进行热处理后，使用Guava自噬检测试剂盒对PBMC进行绝对定量，并与未处理的对照进行比较。该方法基于抗体标记，随后的流式细胞术分析能够通过测量FITC强度来确定自噬体的数量。本方案提供了快速、可靠的结果，不仅可用于研究热疗，还可用于研究其他干预措施，如热量限制。关键特性 • 对活细胞中的自噬进行快速可靠的离体定量。 • 用于测定原代人血细胞自噬的优化方案。 • 允许测试与自噬相关的活性物质和治疗方法。 • 基于流式细胞术的自噬测定方法。
