The limited availability of rapid and reliable flow cytometry-based assays for ex vivo quantification of autophagy has hampered their clinical applications for studies of diseases pathogenesis or for the implementation of autophagy-targeting therapies. To this aim, we modified and improved the protocol of a commercial kit developed for quantifying the microtubule-associated protein 1A/1B light chain 3B (LC3), the most reliable marker for autophagosomes currently available. The protocol modifications were set up measuring the autophagic flux in neoplastic (THP-1 cells) and primary cells (peripheral blood mononuclear cells; PBMC) of healthy donors. Moreover, PBMC of active tuberculosis (TB) patients were stimulated with the Mycobacterium tuberculosis purified protein derivatives or infected with live Mycobacterium bovis bacillus Calmette-Guerin (BCG). We found that the baseline median fluorescent intensity (MFI) of THP-1 cells changed depending on the time of sample acquisition to the flow cytometer. To solve this problem, a fixation step was introduced in different stages of the assay's protocol, obtaining more reproducible and sensitive results when a post-LC3 staining fixation was performed, in either THP1 or PBMC. Furthermore, since we found that results are influenced by the type and the dose of the lysosome inhibitor used, the best dose of Chloroquine for LC3 accumulation were set up in either THP-1 cells or PBMC. Finally, applying these experimental settings, we measured the autophagic flux in CD14+ cells from active TB patients' PBMC upon BCG infection. In conclusion, our data indicate that the protocol modifications here described in this work improve the stability and accuracy of a flow cytometry-based assay for the evaluation of autophagy, thus assuring more standardised cell analyses.

中文翻译：

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通过流式细胞术优化人类细胞系和原代细胞中自噬的测量。

用于自噬的离体定量的基于快速和可靠的基于流式细胞术的测定方法的有限可用性已经阻碍了其在疾病发病机理研究或自噬靶向疗法的实施中的临床应用。为此，我们修改和改进了开发用于量化微管相关蛋白1A / 1B轻链3B（LC3）的商业试剂盒的协议，该蛋白是目前可用的自噬体的最可靠标记。建立协议修改以测量健康供体的肿瘤细胞（THP-1细胞）和原代细胞（外周血单核细胞； PBMC）中的自噬通量。此外，用结核分枝杆菌纯化的蛋白衍生物刺激活动性结核病（TB）患者的PBMC或感染活的牛分枝杆菌Calmette-Guerin（BCG）。我们发现，THP-1细胞的基线中值荧光强度（MFI）随流式细胞仪采集样品的时间而变化。为了解决这个问题，在检测方案的不同阶段引入了固定步骤，当在THP1或PBMC中进行LC3后染色固定时，可获得更多的可重复和敏感的结果。此外，由于我们发现结果受溶酶体抑制剂类型和剂量的影响，因此在THP-1细胞或PBMC中设置了用于LC3积累的最佳氯喹剂量。最后，应用这些实验设置，我们测量了BCG感染后活动性结核病患者PBMC CD14 +细胞的自噬通量。总之，我们的数据表明，本文中描述的方案修改可改善基于流式细胞术的自噬评估方法的稳定性和准确性，从而确保进行更为标准化的细胞分析。