Monitoring Autophagic Activity In Vitro and In Vivo Using the GFP-LC3-RFP-LC3ΔG Probe

Autophagy is a major degradative pathway influencing various biological processes and diseases. A method for measuring autophagic activity is essential to elucidate the roles and mechanisms of autophagy. Although several relevant methods have been reported, the quantitative monitoring of autophagic activity in cells and in animals remains challenging. This chapter provides methods involving the recently developed autophagic flux probe GFP-LC3-RFP-LC3ΔG. This probe is cleaved to produce GFP-LC3 and RFP-LC3ΔG, the former of which is degraded by autophagy, and the latter of which is maintained in the cytoplasm to serve as an internal control. Autophagic activity can be simply and quantitatively estimated by determining the ratio of GFP and RFP signal intensities. The probe is appropriate for cell cultures as well as in vivo analyses. In this chapter, we describe the utility of this probe for measuring autophagic activity in cultured cells by flow cytometry and for analyzing autophagic activity in zebrafish tissues by confocal microscopy.