自噬溶酶体系统（ALS）对于细胞稳态至关重要，有助于维持整个身体的健康，并且与诸如癌症或心血管疾病等疾病有关。为了确定自噬通量，在体内抑制溶酶体降解是必要的，这极大地复杂了自噬的测量。因此，在本研究中，血液细胞被用作研究对象，因为它们易于分离和常规操作。在本研究中，我们提供了详细的协议，以确定从人类和老鼠全血中分离的外周血单个核细胞中（PBMCs）的自噬通量，并广泛讨论了两种方法的优缺点，这在我们所知道的情况下是首次。 PBMCs 的隔离使用密度梯度离心法进行。为了最大程度地减少实验条件对自噬通量的影响，细胞在其血清中直接用 concanamycin A（ConA）处理2小时，温度为37°C，对于老鼠细胞则在填充有NaCl的血清中进行。ConA 处理会降低老鼠 PBMCs 的溶酶体蛋白酶活性，并增加 Sequestosome 1（SQSTM1）蛋白和LC3A/ B-II：LC3A/B-I比率，而转录因子EB则未发生改变。老化进一步增强了 ConA 相关的 SQSTM1 蛋白在老鼠 PBMCs 中的增加，但心肌细胞中没有，表明自噬通量的组织特异性差异。 在人类 PBMCs 中，ConA 处理也降低了溶酶体活性，并增加了LC3A/B-II 蛋白水平，证明了在人类样本中成功检测到自噬通量。总之，这两种协议都适用于确定老鼠和人类样本中的自噬通量，并可能有助于更好地理解衰老和疾病模型中自噬失调的机制，并进一步开发新的治疗策略。版权所有©2023 Walter、Jung、Herpich、Norman、Pivovarova-Ramich 和 Ott。

The autophagy lysosomal system (ALS) is crucial for cellular homeostasis, contributing to maintain whole body health and alterations are associated with diseases like cancer or cardiovascular diseases. For determining the autophagic flux, inhibition of lysosomal degradation is mandatory, highly complicating autophagy measurement in vivo. To overcome this, herein blood cells were used as they are easy and routinely to isolate. Within this study we provide detailed protocols for determination of the autophagic flux in peripheral blood mononuclear cells (PBMCs) isolated from human and, to our knowledge the first time, also from murine whole blood, extensively discussing advantages and disadvantages of both methods. Isolation of PBMCs was performed using density gradient centrifugation. To minimize changes on the autophagic flux through experimental conditions, cells were directly treated with concanamycin A (ConA) for 2 h at 37°C in their serum or for murine cells in serum filled up with NaCl. ConA treatment decreased lysosomal cathepsins activity and increased Sequestosome 1 (SQSTM1) protein and LC3A/B-II:LC3A/B-I ratio in murine PBMCs, while transcription factor EB was not altered yet. Aging further enhanced ConA-associated increase in SQSTM1 protein in murine PBMCs but not in cardiomyocytes, indicating tissue-specific differences in autophagic flux. In human PBMCs, ConA treatment also decreased lysosomal activity and increased LC3A/B-II protein levels, demonstrating successful autophagic flux detection in human subjects. In summary, both protocols are suitable to determine the autophagic flux in murine and human samples and may facilitate a better mechanistic understanding of altered autophagy in aging and disease models and to further develop novel treatment strategies.Copyright © 2023 Walter, Jung, Herpich, Norman, Pivovarova-Ramich and Ott.