Flow cytometric assays for the study of autophagy.

ABSTRACT

The use of flow cytometry to study the autophagic process has recently led to the development of numerous assays measuring various aspects of the autophagic process. These include the detection of the autophagy marker, the microtubule associated protein LC3B, cell cycle analysis of LC3B expression, increase in lysosomal mass, as well as organelle specific autophagy and the measurement of mitochondrial function. We employed a range of autophagy inducing agents to determine the degree of LC3B up-regulation and corresponding cell cycle distribution, increase in lysosomal mass and mitochondrial dysfunction, as well as the relative preference for the specific type of microautophagy or organelle phagy. A variety of autophagy inducing agents were compared these included rapamycin, chloroquine, various nutrient starvation treatments on two cell types, Jurkat T-cell leukaemia and K562 erythromyeloid leukaemia cell lines. Given that numerous autophagy inducing agents cause cell cycle arrest, the cell cycle phase distribution was investigated and LC3B antigen was shown to increase as cells progressed through the cell cycle. LysoTracker dyes have been previously employed to investigate the autophagic process and here the LysoTracker signal increased in autophagic cells in relation to controls. Organelle autophagy of mitochondria and Endoplasmic Reticulum (ER), termed mitophagy and ER phagy was determined flow cytometrically by the employment of organelle mass probes, MitoTracker Green (MTG) and ER Tracker Green (ERTG). A modification of the cell cycle analysis width and area analysis employed for DNA content determinations was developed to show changes in organelle mass on a linear scale. Relative changes in linear scaled median fluorescence intensity (MFI) was compared to control cells to determine the degree and type of organelle phagy induced by a range of autophagy inducing agents and treatments. These flow cytometric organelle phagy and lysosome mass assays can be used by researchers to study the autophagic process further in terms of cell and mitochondrial functionality over time in a cell dependent manner as an adjunct to LC3B measurements.