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Autophagy Analysis Using Object Spot CountingDownload
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February 05, 2018
Authors: Sarah Beckman, BioTek Instruments, Inc., Winooski, VT USA; Nadia Rana and Wini Luty, Enzo Life Sciences, Farmingdale, NY USA
A continuous supply of nutrients is required in a developing organism 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. Starvation, stress, or reduced availability of growth factors induce eukaryotic cells to adjust their metabolism in order to survive. One of the key responses to such a stress is autophagy. Autophagy is a highly conserved process by which cells break down their intracellular components, which among other things, aids in maintaining the amino acid pool during starvation. Autophagy is critical for the maintenance of cellular homeostasis. However, dysregulated autophagy can lead to death of healthy cells and survival of cancerous ones.
Traditional methods of autophagy analysis include electron microscopy and western blot analysis of autophagy associated proteins. Electron microscopy is limited by the necessity of specialized expertise, and open to interpretation when identifying autophagosome structures. Flow cytometry or western blot measurements of autophagy associated proteins, such as LC3-II, do not always correlate with formation of autophagosomes and do not give per-cell numbers of autophagosomes.
Spot counting analysis using manual methods is tedious, time consuming, and prone to error. In contrast, automated image analysis consistently and precisely determines the number of autophagy positive spots per nuclei across multiple samples. This rapid process saves time from manual counting and applies the same set of parameters to all of the samples, lessening the user bias which is inevitable in manual counting. Here we present the use of an autophagy dye in combination with automated objectbased spot counting to quantitatively assess the effects of known autophagy activators starvation and rapamycin on cellular autophagy. We perform automated object based spot counting analysis which allows for the determination of size and number of autophagosomes per cell.