Background

Autophagy is a highly conserved cellular process that removes unwanted or damaged cellular components [1,2]. Although first described as a mechanism to protect cells against starvation [3], it was later understood that autophagy has an important role in non-starving cells to maintain cellular homeostasis [4,5]. Currently, autophagy is recognized as being involved in organ development [6,7] and in coping with infections [8], environmental stressors [9], and aging [10]. It is also known that extreme cellular stress or insufficient autophagic response leads to the deterioration of cellular functions, possibly leading to cell death [11–14].

There are a number of different tools available to study autophagy [15], including electron microscopy and measurement of Atg8-family proteins [e.g., microtubule-associated protein 1A/1B-light chain 3 (LC3)] by various techniques including western blot, flow cytometry, immunofluorescent staining, or fluorescent microscopy, providing an assessment of autophagic activity. Autophagic proteins exhibit tissue- and cell-specific expression and different turnover rates. In addition, some of these approaches require cell fixation, permeabilization, or transfection of exogeneous fluorescent proteins. Since these procedures can alter the physiological expression of autophagy markers, they are not entirely suitable for the accurate assessment of autophagic flux (the rate of autophagosome formation over time, an indicator of autophagic activity) per se.

Live cell imaging is a valuable tool for studying biological processes. When combined with suitable fluorescent marker(s), it allows the visualization and quantification of complex biological processes, such as autophagy, in real time, even at single-cell resolution. Recently, a novel fluorescent dye, DAPRed, was developed for autophagy studies [16]. DAPRed is a small fluorescent molecule that is incorporated into the autophagosome membrane during the double membrane formation [16]. DAPRed allows the real-time visualization of autophagosomes (i.e., formation and subcellular tracking of autophagosomes), enabling autophagic flux measurement in living cells without the need for complex molecular techniques such as cloning or transduction. When DAPRed is combined with adequate imaging techniques, such as confocal or super resolution microscopy, it allows the study of autophagy kinetics and the interactions with other cellular processes (e.g., lysosomal degradation [17]). DAPRed has already been used in semiquantitative studies to address the activation of autophagy [18–20] or autophagic flux measurement [21]. Here, we describe a procedure for the measurement of autophagic flux in single live cells using DAPRed. This procedure involves cell stimulation with the positive control Rapamycin to induce autophagy and marking the plasma membrane with a fluorescently tagged tomato lectin for high spatial precision for single-cell DAPRed fluorescence detection. Autophagy is a dynamic biological process in which autophagosomes are generated and consumed upon merging with lysosomes. Autophagic activity is best described by autophagic flux, which is the rate of autophagosome generation over time. In this protocol, we explain the importance of inhibiting autophagosome–lysosome fusion and the conditions for correctly determining autophagic flux.