Easy Detection of Mitophagy in Neuronal Cell Lines using Mitophagy Detection Kit

What is Mitophagy?

Mitophagy, which is the selective degradation of mitochondria by autophagy, is one of the important cellular processes that are responsible for breaking down cellular contents. While Mitochondria are one of the cytoplasmic organelles that play a crucial role in cells such as production of energy for cell viability, mitochondria are an important source of ROS (reactive oxygen species). This ROS production contributes to mitochondrial damage and the damaged mitochondria is removed by mitophagy.

Why neuronal cell lines?

In 2008, Dr. Youle et al. reported that Parkin promotes autophagy of damaged mitochondria. A human neuroblastoma cell line SH-SY5Y has high level of endogenous Parkin in the cells. SH-SY5Y is often used for mitophagy studies.
Recently, Dr. Fang et al. succeeded in detecting mitophagy in SH-SY5Y and primary rat cerebral cortical neurons with Dojindo’s Mitophagy Detection Kit.

Additional paper information
Utilized cell line:SH-SY5Y and primary rat cerebral cortical neurons E. Fang, et al., "Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway", Scientific Reports, 2017, 7, 46208.

How does Mitophagy Detection Kit work?

Dojindo’s Mitophagy Detection Kit is a unique product that can detect mitophagy by just adding one reagent (Mtphagy Dye). Mtphagy Dye accumulates in the intact mitochondria and the dye is immobilized with chemical bond. Once depolarization occurs and mitophagy is induced, the damaged mitochondria fuses to lysosome and then Mtphagy Dye emits a high fluorescence.

Technical Tips for Successful Assay

  1. Use the cells which have high level of endogenous Parkin like SH-SY5Y.
  2. Add Mtphagy Dye working solution BEFORE inducing mitophagy. (Mtphagy Dye accumulates in the intact mitochondria).
  3. Use Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) or Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for the positive control.
    *FCCP or CCCP concentration: 10 µM - 30 µM, incubation time at 37℃ 3hr - 24 hr
  4. Use appropriate filter : Mtphagy Dye : Ex.500~560 nm, Em.670~730 nm for microscopy detection.

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