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.
FCM
Mitochondrial contribution to lipofuscin formation
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.
Age-associated changes in human CD4+ T cells point to mitochondrial dysfunction consequent to impaired autophagy
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).
Microplate Reader
PINK1 depletion sensitizes non-small cell lung cancer to glycolytic inhibitor 3-bromopyruvate: involvement of ROS and mitophagy
No. | Sample | Instruments | Publications |
1) | Cell(HeLa, MEF) | Fluorescencemicroscope | H. Iwashita, H. T. Sakurai, N. Nagahora, M. Ishiyama, K. Shioji, K. Sasamoto, K. Okuma, S. Shimizu, and Y. Ueno, “Small fluorescent molecules for monitoring autophagic flux.”, FEBS Letters., 2018, 592, (4), 559–567. |
2) | Cell(HeLa) | Fluorescencemicroscope | T. Sakata, A. Saito and H. Sugimoto, “In situ measurement of autophagy under nutrient starvation based on interfacial pH sensing.”, Scientific Reports., 2018, 8, 8282. |
3) | Cell(HS-MM) | Fluorescencemicroscope | Y. Egawa, C. Saigo, Y. Kito, T. Moriki and T. Takeuchi , “Therapeutic potential of CPI-613 for targeting tumorous mitochondrial energy metabolism and inhibiting autophagy in clear cell sarcoma.”, PLoS One., 2018, 13, (6), e0198940. |
4) | Cell(HaCaT) | Fluorescencemicroscope | S. Abe, S. Hirose, M. Nishitani, I. Yoshida, M. Tsukayama, A. Tsuji and K. Yuasa , “Citrus peel polymethoxyflavones, sudachitin and nobiletin, induce distinct cellular responses in human keratinocyte HaCaT cells.“, Biosci. Biotechnol. Biochem. ., 2018, 82, (12), 1347. |
5) | Cell(KGN) | Fluorescencemicroscope | W. Yuping, M. Congshun, Z. Huihui, Z. Yuxia, C. Zhenguo and W. Liping, “Alleviation of endoplasmic reticulum stress protects against cisplatin-induced ovarian damage.”, Reprod. Biol. Endocrinol., 2018,doi: 10.1186/s12958-018-0404-4. |
6) | Cell(BmN) | Fluorescencemicroscope | S. Xue, F. Mao, D. Hu, H. Yan, J. Lei, E. Obeng, Y. Zhou, Y. Quan, and W. Yu, “Acetylation of BmAtg8 inhibits starvation-induced autophagy initiation.”, Mol. Cell Biochem., 2019,doi: 10.1007/s11010-019-03513-y. |
7) | Cell(HeLa) | Fluorescence microscope | F. Hongbao,Y. Shankun, C. Qixin, L. Chunyan, C. Yuqi, G. Shanshan, B. Yang, T. Zhiqi, L. Z. Amanda, T. Takanori, C.Yuncong, G. Zijian, H. Weijiang and D. Jiajie , “De Novo-Designed Near-Infrared Nanoaggregates for Super-Resolution Monitoring of Lysosomes in Cells, in Whole Organoids, and in Vivo.”, ACS Nano, 2019, 13, (12), 1446. |
8) | Cell(RT-7) | FlowCytometer | E. Sasabe, A. Tomomura, N. Kitamura and T. Yamamoto, “Metal nanoparticles-induced activation of NLRP3 inflammasome in human oral keratinocytes is a possible mechanism of oral lichenoid lesions.”, Toxicol In Vitro., 2020, 62, 104663. |
9) | Cell(multiple myeloma) | Fluorescence microscope | J. Xia, Y. He, B. Meng, S. Chen, J. Zhang, X. Wu, Y. Zhu, Y. Shen, X. Feng, Y. Guan, C. Kuang, J. Guo, Q. Lei, Y. Wu, G. An, G. Li, L. Qiu, F. Zhan and W. Zhou, “NEK2 induces autophagy-mediated bortezomib resistance by stabilizing Beclin-1 in multiple myeloma.”, Mol Oncol, 2020, DOI: 10.1002/1878-0261.12641. |
10) | Cell(Human L2) | Fluorescence microscope | Q. Xu, W. Shi, P. Lv, W. Meng, G. Mao, C. Gong, Y. Chen, Y. Wei, X. He, J. Zhao, H. Han, M. Sun and K. Xiao, “Critical role of caveolin-1 in aflatoxin B1-induced hepatotoxicity via the regulation of oxidation and autophagy.”, Cell Death Dis., 2020, 11(1), 6. |
11) | Cell(Cardiomyocytes) | Fluorescence microscope | L Cui, LP Zhao, JY Ye, L Yang, Y Huang, X.P. Jiang, Q. Zhang, JZ. Jia, DX. Zhang and Y. Huang, “The Lysosomal Membrane Protein Lamp2 Alleviates Lysosomal Cell Death by Promoting Autophagic Flux in Ischemic Cardiomyocytes.“, Front Cell Dev Biol, 2020,DOI:10.3389/fcell.2020.00031. |
12) | Cell(IPEC-J2) | Fluorescence microscope | Y Yang, J Huang, J Li, H Yang and Y. Yin, “The Effects of Butyric Acid on the Differentiation, Proliferation, Apoptosis, and Autophagy of IPEC-J2 Cells..”, Curr. Mol. Med., 2020, 20(4), 307. |
13) | Cell(Fibroblasts, Kidney epithelial cells) | Fluorescence microscope | M. M. Ivanova, J. Dao, N. Kasaci, B. Adewale, J. Fikry and O. G. Alpan , “Rapid Clathrin-Mediated Uptake of Recombinant α-Gal-A to Lysosome Activates Autophagy”, Biomolecules , 2020, 10(6), 837. |
14) | Cell(NHEKs) | Fluorescence microscope | S. Ikeoka and A. Kiso , “The Involvement of Mitophagy in the Prevention of UV-B-Induced Damage in Human Epidermal Keratinocytes “, J. Soc. Cosmet. Chem. Jpn., 2020, 54(3), 252. |
Related Categories Cell Staining Intracellular Fluorescent Probes Mitochondria Research