Kit content ：
Mtphagy Dye 5 μg x 1, Lyso Dye 30 μg x 1
Storage Condition ：
Store at 0-5o
C and protect from light.
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 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.
Excitation and emission spectra of Mtphagy Dye and Lyso Dye
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 37o
C overnight in a 5%-CO2
incubator. The cells were transfected with Parkin plasmid vector by HilyMax transfection reagent from Dojindo (Code#:H357), and incubated at 37o
C overnight. The Parkin expressed HeLa cells were washed with Hanks’ HEPES buffer twice and then incubated at 37o
C for 30 minutes with 250 μl of 100 nmol/l Mtphagy Dye working solution. 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 37o
C for 30 minutes. The cells were washed with Hanks’ HEPES buffer twice and then co-localization of Mtphagy and Lyso Dye was observed.