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.
Procedure (Adherent cell):
1.Cells were washed twice with DMEM and afterwards incubated at 37 °C for 30 min with 100 nmol Mtphagy Dye diluted in DMEM.
2.After this incubation cells were again washed twice with DMEM followed by the addition of complete DMEM.
3.The induction of mitophagy was then accomplished by the addition of 20 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 24 h. Subsequently, fibroblasts were trypsinized and fluorescence intensity of Mtphagy Dye was measured by flow cytometry at 488 nm excitation and 655–730 nm emission.
Procedure (Suspension cell):
- Briefly, 2×106 CD4+ T cells were divided into four tubes containing serum-free RPMI.
- Tube #1 was for unstained cells that followed all washing and media changing processes.
- 100 nmol/l Mtphagy Dye working solution was added to tubes #2, 3, 4 and then all tubes were incubated at 37°C for 30 minutes.
- The cells were then washed with serum-free medium. After discarding the supernatant, complete medium (RPMI 1640, 10% FBS, 1% P/S/G) was added to all the tubes.
- Ten μmol/l CCCP (Sigma-Aldrich) mitophagy-inducer was then added to tube #3, 100 nmol/l Bafilomycin A1 (Sigma-Aldrich) autophagy inhibitor was added to tube #4.
- All tubes were then incubated at 37 °C for 18 hours.
- After 18 hours, cells were washed with FACS buffer.
- Mtphagy Dye fluorescence detection by flow cytometry (BD FACSCANTO II) was performed using 488 nm for excitation and 695 nm for emission (this corresponds to the PerCP cy5.5 channel). Data were analyzed using FLOWJO software (version 10).
2. 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.
3. 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.
4. Y. Feng, et al. “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”, British Journal of Pharmacology, 2017, doi: 10.1111/bph.14033 .
5. Kazuhisa Kameyama, “Induction of mitophagy-mediated antitumor activity with folate-appended methyl-β-cyclodextrin”, International Journal of Nanomedicine., 2017, 12, 3433-3446.
6. 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.
7. 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.2018.02.149.8)
8. N. Furuya, S. Kakuta, K. Sumiyoshi, M. Ando, R. Nonaka, A. Suzuki, S. Kazuno, S. Saiki and N. Hattori, “NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy.”, EMBO Rep., 2018, doi: 10.15252/embr.201846363.
9. K. Araki, K. Kawauchi, W. Sugimoto, D. Tsuda, H. Oda, R. Yoshida, K. Ohtani, Mitochondrial protein E2F3d, a distinctive E2F3 product, mediates hypoxia-induced mitophagy in cancer cells, Communications Biology, 2019, doi: 10.1038/s42003-018-0246-9
10. E. Adegoke, S. Adeniran, Y. Zeng, X. Wang, H. Wang, C. Wang, H. Zhang, P. Zheng and G. Zhang , “Pharmacological inhibition of TLR4/NF-κB with TLR4-IN-C34 attenuated microcystin-leucine arginine toxicity in bovine Sertoli cells.”, J Appl Toxicol., 2019,doi: 10.1002/jat.3771.
11. E. F. Fang, Y. Hou, K. Palikaras, B. A. Adriaanse, J. S. Kerr, B. Yang, S. Lautrup, M. M. Hasan-Olive, D. Caponio, X. Dan, P. Rocktaschel, D. L. Croteau, M. Akbari, N. H. Greig, T. Fladby, H. Nilsen, M. Z. Cader, M. P. Mattson, N. Tavernarakis and V. A. Bohr, “Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease.”, Nat. Neurosci. ., 2019,DOI:10.1038/s41593-018-0332-9.
12. Takushi Namba, “BAP31 regulates mitochondrial function via interaction with Tom40 within ER-mitochondria contact sites”, Science Advances, 2019, DOI: 10.1126/sciadv.aaw1386.