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Directly Quantifying Autophagy in Peripheral Blood Mononuclear Cells (PBMCs).

Autophagy assays involving stably transfected LC3-GFP cell lines can be subjective, may lead to false positives due to GFP-LC3 aggregation and are not suitable for analysis of clinical specimens. Counting GFP-LC3 punctae only monitors increases in autophagosomes, which may represent pathway blockage and is not sufficient for determining autophagic flux. Dr. Anna Katharina Simon of the University of Oxford and colleagues developed a high throughput, statistically robust technique to quantify autophagy in PBMCs using an imaging flow cytometer1. The method quantifies delivery of LC3 into lysosomes, using fluorescently-labeled LC3 mAb in combination with Enzo’s Lyso-ID® Red detection kit. The log transformed Pearson’s correlation coefficient is employed to calculate the degree of overlapping pixel intensities between different fluorescence imaging channels. Applying the assay to PBMCs from young and old healthy donors highlighted a significant decrease in basal and starvation-induced autophagy in older adults. The assay should facilitate investigation of autophagy in lymphocytes and other leukocytes. Even in inherited diseases not directly affecting the hematopoietic system, PBMCs can accurately reflect autophagy status of the affected tissue, as demonstrated for distal hereditary motor neuropathy type II2.

Enzo Life Sciences offers an unrivaled portfolio of tools for autophagy research, including live cell analysis kits, immunoassays, compound libraries and antibodies, some of which are briefly described below.

 

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