Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry

  • Tonino Alonzi Translational Research Unit, Department of Epidemiology and Preclinical Research, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, Rome, Italy. https://orcid.org/0000-0002-7706-5828
  • Elisa Petruccioli Translational Research Unit, Department of Epidemiology and Preclinical Research, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, Rome, Italy. https://orcid.org/0000-0003-3418-6803
  • Valentina Vanini Translational Research Unit, Department of Epidemiology and Preclinical Research, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, Rome, Italy. https://orcid.org/0000-0001-8476-964X
  • Gian Maria Fimia Translational Research Unit, Department of Epidemiology and Preclinical Research, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, Rome; Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. https://orcid.org/0000-0003-4438-3325
  • Delia Goletti | delia.goletti@inmi.it Translational Research Unit, Department of Epidemiology and Preclinical Research, National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS, Rome, Italy. https://orcid.org/0000-0001-8360-4376

Abstract

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.

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Published
2019-06-26
Section
Original Papers
Supporting Agencies
National Institute of Health of USA (NIH 1R21AI127133-01), Italian Ministry of Health (“Ricerca Finalizzata”: RF-GR54 and “Ricerca Corrente”), Italian Ministry of University and Research - PRIN 2015 No. 20152CB22L, European Union (643381-TBVAC2020-H2020-PHC-2014-2015)
Keywords:
Autophagy, tuberculosis, BCG, flow cytometry, LC3, PBMC, THP-1
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How to Cite
Alonzi, T., Petruccioli, E., Vanini, V., Fimia, G. M., & Goletti, D. (2019). Optimization of the autophagy measurement in a human cell line and primary cells by flow cytometry. European Journal of Histochemistry, 63(2). https://doi.org/10.4081/ejh.2019.3044