Sarah Beckman Ph.D.¹, Wini Luty^2
1BioTek Instruments, Inc., Winooski, Vermont, USA,
²Enzo Life Sciences, Farmingdale, New York, USA


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INTRODUCTION

A constant supply of nutrients is required during development to provide the energy necessary for growth, metabolism, and survival. Eukaryotic cells have evolved a variety of mechanisms to adjust their metabolic activities in response to changes in nutrient levels. Nutrient starvation, stress, or reduced availability of growth factors induces eukaryotic cells to adjust their metabolism in order to survive. One of the key responses to such a stress is autophagy.

Autophagy or “self-eating” is a highly conserved process by which cells break down their intracellular components. In a healthy cell under physiological conditions, autophagy is protective. In fact, autophagy plays a variety of important roles, including the maintenance of the amino acid pool during starvation, damaged protein and organelle turnover, prevention of neurodegeneration, tumor suppression, cellular differentiation, clearance of intracellular microbes, and regulation of innate and adaptive immunity.

The first step of autophagy is formation of the phagophore, a cup-shaped double membrane. The edges of this membrane elongate and engulf portions of the cytoplasm, including intracellular material such as damaged organelles and misfolded proteins. The isolation membrane expands and its open ends fuse to form a double-membrane structure called the autophagosome. Autophagosomes then fuse with lysosomes to form autolysosomes and the contents inside the autophagosome are degraded by lysosomal hydrolases. The intracellular material is then recycled back into the cytosol.

One of the most well-known inducers of autophagy is starvation. Through autophagy, amino acids and other nutrients are recycled from long-lived proteins, organelles, and other components of the cytoplasm, providing an internal reserve of nutrients. Starvation rapidly induces autophagy, in part by inactivation of the mTOR (mammalian target of rapamycin) substrate S6K.6 In a nutrient-rich environment, mTOR inhibitors such as rapamycin can induce autophagy.

Autophagy plays a role in both the pathogenesis and prevention of disease. This is especially true in cancer, where elimination of damaged intracellular components through autophagy suppresses tissue injury and tumor initiation. However, in an established tumor, autophagy promotes cancer progression by providing substrates for metabolism, maintaining functional mitochondria, and fostering survival.

Traditional methods of autophagy analysis include electron microscopy and western blot analysis of LC3- II. Electron microscopy is limited by the necessity of specialized expertise, and open when identifying an autophagosome structure. Furthermore, flow cytometry or western blot measurements of LC3-II do not always correlate with formation of autophagosomes and do not give per-cell numbers of autophagosomes. In this application note, we describe the process of using CYTO-ID® Autophagy Detection Dye combined with Gen5 3.03 software to analyze the effects of serum starvation and rapamycin on autophagosome number in HeLa cells. We perform analysis with Gen5 3.03 with object spot counting capability, which allows us to determine the number of autophagosomes per cell as well as their size.