Mitochondria Research for Mammalian Cells
∼ Feature ∼
Mtphagy Dye (Item# MT02) ： Mtphagy Dye 5 μg x 3
Storage Condition ： Store at 0-5 oC and protect from light.
Lyso Dye stain lysosome and is used to confirm the fusion of Mtphagy Dye-labeled mitochondria and lysosome. Therefore, we strongly recommend using Mitophagy Detection Kit (MD01) for First Time Users.
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.
Transfection procedure is not needed for mitophagy imaging.
Comparison with Autophagy Marker
CCCP(carbonyl cyanide m-chlorophenyl hydrazone) has been added to normal and Parkin expressed cells. The strong fluorescence was not observed in normal HeLa cells(A)(B). On the other hand, the trong fluorescence was shown in Parkin expressed cells in 18 hours after additon of CCCP(C). Some of the puncta are co-localized with the autophagy marker(GFP-LC3). In addition, suppressed fluorescence of Mtphagy dye was observed when autophagy inhibitor, bafilomycin was added to Parking expressed cells(D). Because lysosomal pH was increased by the additon of bafilomycin.
Mitophagy Detection in Starved Cells
Living HeLa cells are co-stained with Mtphagy Dye and Lyso Dye under mitophagy induced condition.
Mtphagy Dye was added to HeLa cells and the cells were incubated for 6 hours under starved condition. Before observation of the cells, lysosomal staining dye, Lyso Dye was added to HeLa cells. The fluorescent intensity of Mtphagy Dye was increased in the starved HeLa cells but not in normal cells. In addition, Mtphagy Dye was co-localized with Lyso Dye in the starved cells.
Experimental Example: Induction of Mitophagy by CCCP
[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)
Mitophagy Induction and Detection of Mitochondrial Membrane Potential Changes
Transfection of Parkin plasmid to HeLa cells
Detection of Mitophagy
Detection of Mitochondrial Membrane Potential
1) 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.
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.
What is an advantage of the kit in comparison to Keima-Red?
Can I use Mitophagy Detection Kit with fixed cells?
Why it is not allowed to mix Mtphagy dye and Lyso dye together?
How many times can I assay with Mitophagy Detection Kit?
Can I store the working solution after it has been prepared?
How long is DMSO stock solution stable?
What are some technical tips for successful assay?
Is the serum in the medium susceptible to live cell imaging?
What is an effect of Phenol Red?
What is the recommended filter?
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: 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: 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.
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: What are some technical tips for successful assay?
1. Add Mtphagy Dye working solution BEFORE inducing mitophagy (Mtphagy Dye accumulates in the intact mitochondria)2. Use appropriate filter - Mtphagy Dye: Ex.500-560 nm, Em.670-730 nm for microscopy detection3. Use CCCP or FCCP for the positive control *FCCP or CCCP concentration: 10 uM-30 uM, incubation time at 37℃ for 3 hours FCCP: Carbonyl cyanide 4-(triguoromethoxy)phenylhydrazone CCCP: Carbonyl cyanide 3-chlorophenylhydrazone4. Use Confocal Microscope if available
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: What is an effect of Phenol Red?
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