Mtphagy DyeMT02 Product Manual

Mitochondria are one of the cytoplasmic organelles that play 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.
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 fuse to lysosome and then Mtphagy Dye emits a high fluorescence. To confirm the fusion of Mtphagy Dye–labeled mitochondria and lysosome, please choose Mitophagy Detection Kit (Dojindo code: MD01) which includes lysosome staining dye, Lyso Dye and Mtphagy Dye.

Figure 1 The mechanism of mitophagy detection with Mtphagy Dye

Mtphagy Dye

5 μg x 3

Store at 0-5 ^oC and protect from light.

Preparation of 100 μmol/l Mtphagy Dye DMSO stock solution

Add 50 μl of DMSO to a tube of Mtphagy Dye (5 μg) and dissolve it with pipetting.

• Store reconstituted DMSO solution at -20 ^oC. The reconstituted solution is stable at -20 ^oC for 1 month.

 

Preparation of 100 nmol/l Mtphagy Dye working solution

Dilute the 100 μmol/l Mtphagy Dye DMSO stock solution with Hanks’ HEPES buffer or serum-free medium to prepare 100 nmol/l Mtphagy working solution.

• Use Hanks’ HEPES buffer or serum-free medium to the dilution because serum in medium is interference with Mtphagy Dye.

• Mtphagy Dye can not be applied to the fixed cells. If the cells are fixed before staining, Mtphagy Dye is unable to accumulate in mitochondria. In addition, the fixation after Mtphagy Dye staining is not recommended because lysosome merged phagosome is not maintained under the acidic condition and consequently the fluorescent intensity of Mtphagy Dye is not increased.

 

Mitophagy detection

1. Prepare cells on dish for assay.
2. Discard the culture medium and wash the cells with Hanks’ HEPES buffer or serum-free medium twice.
3. Add an appropriate volume of 100 nmol/l Mtphagy Dye working solution and then incubate at 37 ^oC for 30　minutes.
4. Discard the supernatant and wash the cells with Hanks’ HEPES buffer or serum-free medium twice.
5. Add medium containing mitophagy-inducing agent and incubate at 37 ^oC for appropriate time. Confirm the mitophagy phenomenon on a fluorescence microscope.
   • To observe the co-localization of Mtphagy Dye and lysosome, incubate at 37 ^oC for 30 minutes with 1 μmol/l Lyso Dye working solution (Dojindo code: MD01) before the observation of mitophagy.
6. Discard the supernatant, wash the cells with Hanks’ HEPES buffer or serum-free medium twice and observe on a fluorescence microscope.

 

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 (Dojindo 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 (Dojindo 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 (Dojindo code:MD01) 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.

Figure 2 Observation of mitophagy using Parkin expressed HeLa cells (upper panel) and normal HeLa cells (lower panel)

Excitation and emission spectra of Mtphagy Dye

λex：530 nm λem：700 nm ＜Recommended Filter＞ Ex：500-560 nm Em：670-730 nm

Mtphagy Dye is patented.