Kit content ： Mtphagy Dye 5 μg x 1, Lyso Dye 30 μg x 1
Storage Condition ： Store at 0-5oC 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 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.
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.
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 37oC overnight in a 5%-CO2 incubator. The cells were transfected with Parkin plasmid vector by HilyMax transfection reagent (Code#:H357), and incubated at 37oC overnight. The Parkin expressed HeLa cells were washed with Hanks’ HEPES buffer twice and then incubated at 37oC 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 37oC 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)
A, E) Fluorescent images of Mtphagy Dye
B, F) Fluorescent images of Lyso Dye
C, G) Fluorescent images of MitoBright Deep Red
D, H) Co-localized fluorescent images of Mtphagy, Lyso Dye and MitoBright Deep Red
-Mtphagy Dye: 561nm(Ex), LP650nm(Em)
-Lyso Dye：488nm(Ex), 502-554nm(Em)
-MitoBright Deep Red：640nm(Ex), 656-700nm(Em)
1)J. Koniga, C. Otta, M. Hugoa, T. Junga, A. L. Bulteaub, T. Grunea and A. Hohna, "Mitochondrial contribution to lipofuscin formation", Redox Biology, 2017, 11, 673.
2) K. Kameyama, "Induction of mitophagy-mediated antitumor activity with folate-appended methyl-β-cyclodextrin", International Journal of Nanomedicine, 2017, 12, 3433.
3) E. F. Fang, T. B. Waltz, H. Kassahun, Q. Lu, J. S. Kerr, M. Morevati, E. M. Fivenson, B. N. Wollman, K. Marosi, M. A. Wilson, W. B. Iser, D. M. Eckley, Y. Zhang, E. Lehrmann, I. G. Goldberg, M. S. Knudsen, M. P. Mattson, H. Nilsen, V. A. Bohr and K. G. Becker, "Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway", Scientific Reports, 2017, 7, (46208), DOI: 10.1038/srep46208.
4) H. Iwashita, S. Torii, N. Nagahora, M. Ishiyama, K. Shioji, K. Sasamoto, S. Shimizu and K. Okuma, "Live Cell Imaging of Mitochondrial Autophagy with a Novel Fluorescent Small Molecule", ACS Chem. Biol., 2017, 12, (10), 2546.
5) Y. Feng, NB. Madungwe, CV. da Cruz Junho and JC. Bopassa, "Activation of G protein-coupled oestrogen receptor 1 at the onset of reperfusion protects the myocardium against ischemia/reperfusion injury by reducing mitochondrial dysfunction and mitophagy.", Br. J. Pharmacol., 2017, 174, (23), 4329.
6) K. M. Elamin, K. Motoyama, T. Higashi, Y. Yamashita, A. Tokuda and H. Arima, "Dual targeting system by supramolecular complex of folate-conjugated methyl-β-cyclodextrin with adamantane-grafted hyaluronic acid for the treatment of colorectal cancer.", Int. J. Biol. Macromol., 2018, doi: 10.1016/j.ijbiomac.2018.02.149.
7) Zhu L, Xie X, Zhang L, et al. TBK-binding protein 1 regulates IL-15-induced autophagy and NKT cell survival. Nature Communications. 2018;9(1). doi:10.1038/s41467-018-05097-5.
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.
What is an advantage of the kit in comparison to Keima-Red?
What is the recommended filter?
How long is DMSO stock solution stable?
Can I store the working solution after it has been prepared?
Is the serum in the medium susceptible to live cell imaging?
Can I use Mitophagy Detection Kit with fixed cells?
How many times can I assay with Mitophagy Detection Kit?
Why it is not allowed to mix Mtphagy dye and Lyso dye together?
Q : What is an advantage of the kit in comparison to Keima-Red?
A : Our kit uses a small molecular fluorescent probe and allows detection of Mitophagy phenomenon without expressing fluorescent protein.
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
Q : How long is DMSO stock solution stable?
A : Mtphagy Dye DMSO stock solution and Lyso Dye DMSO stock solution are stable for 1 month at -20℃.
Q : Can I store the working solution after it has been prepared?
A : No, since the working solution is unstable, prepare working solution before each experiment and do not store the prepared working solution.
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 : Can I use Mitophagy Detection Kit with fixed cells?
A : No, the kit is suitable for live cells because Mtphagy Dye accumulates in intact mitochondria.
Q : How many times can I assay with Mitophagy Detection Kit?
A : For 96 wells plates format (100 ul/well), the kit is sufficient for 5 plates. For 35 mm dish format (2ml), the kit is sufficient for 25 assays.
Q: Why it is not allowed to mix Mtphagy dye and Lyso dye together?
A: Since fluorescence of Lyso dye fades after long periods of staining, if both dyes are added together, Lyso dye does not emit enough fluorescence for double staining. Hence, Lyso dye is added right before the observation.