Work

High-Throughput Quantitative Detection of Basal Autophagy and Autophagic Flux Using Image Cytometry

Year: 2019

Type: article

Abstract: Quantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevent accurate quantification. We report a high-throughput, inexpensive, reliable, and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining. Methods summary A high-throughput, inexpensive, reliable, and objective method for studying both basal autophagy and autophagic flux is reported. This approach uses acridine orange staining of late-stage autophagy and image cytometry to quantify autophagy.

Source: BioTechniques

DOI: 10.2144/btn-2019-0044

Authors: Gopika SenthilKumar, Justin H. Skiba, Randall J. Kimple

Institutions: University of Wisconsin–Madison, University of Wisconsin Carbone Cancer Center

Language: English

Topic: Autophagy in Disease and Therapy

Subfield: Cell Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,415