Flow Cytometry-Based Assessment of Mitophagy Using MitoTracker

Mitophagy is an important mechanism in mitochondrial quality control through autophagic clearance of damaged mitochondria and has been considered to assume protective roles against some diseases, especially neurodegeneration, including Parkinson's disease (Haelterman et al., 2014), and Alzheimer's disease (Ye et al., 2015). To understand the role of mitophagy in these diseases, measures to assess mitophagy are of great importance. Among those established methods, engulfment of mitochondria by autophagosome shown by transmission electron microscope (TEM) provides unequivocal evidence of occurrence of mitophagy (Klionsky et al., 2012). However, it demands painstaking effort and is difficult to quantify mitophagy. MitoTracker has emerged as a useful tool in evaluating mitophagy. Mitochondrial potential-independent MitoTracker is able to stain live mitochondria and enables demonstration of colocalization of mitochondria and autophagosome or lysosome with the aid of corresponding marker (Klionsky et al., 2012). Alternatively, since MitotTracker Green has been used to represent mitochondrial mass (Agnello et al., 2008; Cottet-Rousselle et al., 2011; Zhou et al., 2011) and a decrease in MitotTracker intensity may indicate the degradation of mitochondria, it has been widely utilized to evaluate mitophagy (Kundu et al., 2008; Valentin-Vega et al., 2012). Recently, a flow cytometry-based approach to determine mitophagy by using MitoTracker Deep Red has been introduced by Mauro-Lizcano et al. (2015). Despite the promising role of MitoTracker, we would like to highlight potential limitations in its interpretation as some factors may significantly affect determination of mitochondrial mass by MitoTracker staining. First, MitoTracker Deep Red is actually a mitochondrial potential-dependent dye though this is usually not highlighted in the manufacturer's manual. It has been used as an index for mitochondrial potential (Lugli et al., 2005; Zhou et al., 2011; Greene et al., 2012). Our data also suggest that the intensity of MitoTracker Deep Red changes along with mitochondrial potential. Mitochondrial depolarization induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment decreased fluorescence intensity of MitoTracker Deep Red, while starvation induced by Earle's Balanced Salt Solution (EBSS) treatment increased fluorescence intensity of MitoTracker Deep Red (Figure 1A). JC-1 assay utilized in our experiments confirmed that CCCP disrupted mitochondrial potential and revealed that EBSS treatment caused mitochondrial potential to increase (Figure 1B), in line with the report that amino acid starvation raised mitochondrial potential (Johnson et al., 2014). Our data and previous reports suggest that MitoTracker Deep Red is a mitochondrial potential-dependent dye and therefore should not be used to quantitatively assess mitophagy. MitoTracker Green has been widely …