February 1, 2017 (Vol. 37, No. 3)

ImmunoChemistry Technologies’ Fluorescent Solution for Autophagy Detection

With the 2016 Nobel Prize in Physiology or Medicine awarded to Yoshinori Ohsumi for his pioneering work in the early 1990s elucidating the genetic basis of the autophagic “self-eating” process, this highly relevant research topic is back in the spotlight!

Using a baker’s yeast model to help explain the genetic basis behind the autophagic processes, he was able to demonstrate this important intracellular contents degradation process in his yeast model and subsequently demonstrate how an analogous process occurs in higher organisms, like humans.

Autophagy is a conserved lysosomal recycling process by which cells break down their own proteins, lipids, and carbohydrates. There are three types of autophagy, including macroautophagy, microautophagy, and chaperone-mediated autophagy. Of the three, macroautophagy (hereafter referred to as autophagy) is the most well-studied of the three processes.

On one hand, autophagy plays a critical role in maintaining cellular homeostasis. This beneficial activity is primarily accomplished by preventing the accumulation of damaged organelles and other miscellaneous intracellular soluble and structural components by means of a disassembly process carried out within the confines of autophagic vacuoles. On the other hand, dysregulation of the autophagy process plays a well-documented role in aging and neurodegenerative disease states such as amyotrophic lateral sclerosis (ALS), Parkinson’s, and Alzheimer’s disease.

Autophagic processes have also been clearly implicated in many types of cancer chemotherapy treatment failures, although its role is paradoxical: having the potential to either induce cell death or promote cell survival.

The complex, dual roles of autophagy are evident when examining cancer metastasis. In some situations, autophagy is thought to inhibit metastasis by restricting necrotic cell death and promoting cell death by autophagy. Alternatively, autophagy may provide cancer cells with an important fitness-enhancing mechanism for coping with cellular stress. Furthermore, autophagy may play different roles during different stages of cancer. During early stages of cancer progression, autophagy is often thought to play a cancer-suppressive role; while during later stages, it is likely promoting metastasis.

Detecting Autophagy

Given the complex array of pathological and physiological roles that autophagy is thought to be involved in, there is an ever-present need for research tools designed for detecting its presence. Autophagy has been commonly detected using a variety of diverse strategies including a GFP/RFP transfection-based approach, a labeled antibody-based approach, or an aliphatic dye-complex based approach.

Both the labeled antibody and transfection-based approaches typically require a cell membrane permeabilization step to facilitate the cell internalization of the autophagy-detection components. Additionally, transfection-based techniques often have a longer workflow involving many additional steps (and thus more opportunities for the introduction of error and subsequent artifacts).

Whole cell analysis using a dye-based approach offers considerable advantages by way of a much shorter workflow protocol, no need for a cell permeabilization step, and no requirement for nonphysiological protein mutations or genetically engineered cell lines. Currently, there are limited options available when pursuing cell-permeant, nontoxic, fluorescent dye autophagy detection strategies.

Historically, a substantial amount of past autophagy research has relied on the use of hydrophobic molecular constructs such as monodansylcadaverine (MDC) for the detection of autophagy events. MDC is an autofluorescent dye that is thought to accumulate in autophagic vacuoles through ion trapping and specific interactions with membrane lipids.

Although capable of being used to detect autophagy, a major drawback for MDC and its related fluorescent analogs is having an excitation requirement in the ultraviolet region. Higher energy (shorter wavelength) ultraviolet-region light is generally incompatible with most living cell systems and often associated with the creation of nonspecific autofluorescence artifacts. Additionally, MDC has a low quantum yield and corresponding low extinction coefficient. These physical properties translate into a need to employ much higher MDC staining concentrations (concentrations around 50–100 µM are common) during autophagy staining protocols. 

ICT’s Autophagy Assay, Red enables researchers to detect and monitor the in vitro development of autophagy in living cells, with the added benefit of working within an excitation/emission spectra that is nonconductive to the generation of autofluorescence artifacts. Autophagy Probe, Red is a cell-permeant aliphatic molecule that fluoresces brightly when inserted into, and subsequently associated with, the lipid membranes of autophagosomes and autolysosomes. Autophagy Probe, Red is easy to use and can be readily detected by flow cytometry (Figure 1).

Figure 1

Kristi Strandberg, Ph.D. ([email protected]), is a staff scientist, Tracy Murphy is director of R&D and quality control, and Brian Lee, Ph.D., is technical consultant, R&D, at ImmunoChemistry Technologies.

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