Mitophagy Detection Kit	Mtphagy Dye

Product Description

Mitophagy Detection Kit is optimized for mammalian cells.

Mitochondria is one of the cytoplasmic organelle that plays a crucial role in cells such as production of energy for cell viability. Recently, Mitophagy appears to be related to Alzheimer and Parkinson disease induced by the accumulation of depolarized mitochondria. Mitophagy serves as a specific elimination system that dysfunctional mitochondria caused by oxidative stress and DNA damage are sequestered into autophagosome, fused to lysosome and degraded by digestion.

This kit is composed of Mtphagy Dye, reagent for detection of mitophagy, and Lyso Dye. Mtphagy Dye accumulates in intact mitochondria, is immobilized on it with chemical bond and exhibits a weak fluorescence from the influence of surrounding condition. When Mitophagy is induced, the damaged mitochondria fuses to lysosome and then Mtphagy Dye emits a high fluorescence. To confirm the fusion of Mtphagy Dye–labeled mitochondria and lysosome, Lyso Dye included in this kit can be used.

E. Fang etc. detected tomatidine induced mitophagy in HeLa cells by using mt-mKeima. Mitophagy was also detected in both primary rat cortical neurons and human SH-SY5Y neural cells using our Mitophagy Detection Kit.³ Mitophagy Detection Kit would be a valid alternative method if protein expression/transfection is not ideal for the experiment.

For more information on Mtphagy Dye compositions and examples, please refer to the publication below:

Iwashita H, Torii S, Nagahora N, Ishiyama M, Shioji K, Sasamoto K, Shimizu S, Okuma K. "Live Cell Imaging of Mitochondrial Autophagy with a Novel Fluorescent Small Molecule."ACS Chem Biol, 2017, doi: 10.1021/acschembio.7b00647.

Notes: Mtphagy Dye and Lyso Dye are Patent Pending.

Simple Procedure

Transfection procedure is not needed for mitophagy imaging.

Comparison with Autophagy Marker

CCCP(carbonyl cyanide m-chlorophenyl hydrazone) has been added to normal and Parkin expressed cells. The strong fluorescence was not observed in normal HeLa cells(A)(B). On the other hand, the trong fluorescence was shown in Parkin expressed cells in 18 hours after additon of CCCP(C). Some of the puncta are co-localized with the autophagy marker(GFP-LC3). In addition, suppressed fluorescence of Mtphagy dye was observed when autophagy inhibitor, bafilomycin was added to Parking expressed cells(D). Because lysosomal pH was increased by the additon of bafilomycin.

Mitophagy Detection in Starved Cells

Living HeLa cells are co-stained with Mtphagy Dye and Lyso Dye under mitophady induced condition.

Mtphagy Dye was added to HeLa cells and the cells were incubated for 6 hours under starved condition. Before observation of the cells, lysosomal staining dye, Lyso Dye was added to HeLa cells. The fluorescent intensity of Mtphagy Dye was increased in the starved HeLa cells but not in normal cells. In addition, Mtphagy Dye was co-localized with Lyso Dye in the starved cells.

Experimental Example: Induction of Mitophagy by CCCP

[Induction of mitophagy by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a mitochondrial-uncoupling reagent with Parkin expressed HeLa cells]

HeLa cells were seeded on μ-slide 8 well (Ibidi) and cultured at 37^oC overnight in a 5%-CO₂ incubator. The cells were transfected with Parkin plasmid vector by HilyMax transfection reagent (Code#:H357), and incubated at 37^oC overnight. The Parkin expressed HeLa cells were washed with Hanks’ HEPES buffer twice and then incubated at 37^oC for 30 minutes with 250 μl of 100 nmol/l Mtphagy Dye working solution containing 100 nmol/l MitoBright Deep Red (Code#:MT08). After the washing of the cells with Hanks’ HEPES buffer twice, the culture medium containing 10 μmol/l CCCP was added to the well. After 24 hours incubation, mitophagy was observed by a fluorescence microscopy. After removing the supernatant, 250 μl of 1 μmol/l Lyso Dye working solution were added to the cells and incubated at 37^oC for 30 minutes. The cells were washed with Hanks’ HEPES buffer twice and then co-localization of Mtphagy, Lyso Dye and MitoBright Deep Red was observed by confocal fluorescence microscopy.

Observation of mitophagy using Parkin expressed HeLa cells (upper panel) and normal HeLa cells (Lower)

Effect of Phenol Red

Figure 1. Background caused by phenol red observed by the fluorescence microscope. However, when using confocal microscope, there is no effect from phenol red.

We strongly recommend using confocal microscope when detecting mitophagy.

Q: Is the serum in the medium susceptible to live cell imaging?

A: Yes, it is susceptible because both Mtphagy Dye and Lyso Dye interfere with the serum in the medium. Please use Hank’s HEPES buffer or serum-free medium.

Q: What is an effect of Phenol Red?

Q: What is the recommended filter?

A: Mtphagy Dye: Ex.500~560 nm, Em.670~730 nm, Lyso Dye: Ex.350~450 nm, Em.500~560 nm