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Mitophagy dye

Manufactured by Dojindo Laboratories
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Sourced in Japan

The Mitophagy dye is a fluorescent probe designed to detect and monitor mitophagy, a selective form of autophagy that targets damaged or dysfunctional mitochondria for degradation. The dye selectively stains mitochondria and can be used to visualize and quantify mitophagy in live cells.

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Lab products found in correlation

Sp8x confocal microscope, by Leica (1 mentions) Lysotracker red, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Mitotracker green, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions) Atg13, by Cell Signaling Technology (1 mentions) Fip200, by Cell Signaling Technology (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions) Mdivi 1, by Beyotime (1 mentions) Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions) Analysis application hybrid cell count, by Keyence (1 mentions) Lysotracker, by Thermo Fisher Scientific (1 mentions) Mitotracker, by Thermo Fisher Scientific (1 mentions) Zen software, by Zeiss (1 mentions)

5 protocols using mitophagy dye

1

Mitophagy Assessment in Cultured Cells

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Viable cells were stained with 100 nmol/l Mitophagy Dye (Dojindo Laboratories) for 30 min and washed in Hank’s Hepes buffer solution. The attached cells were stimulated with 10 μmol/l carbonyl cyanide m-chlorophenyl hydrazone (Nacalai Tesque) for 24 h before observation, as described in the manufacturer’s protocol. Fluorescent images were obtained using the Leica SP8 X confocal microscope.
Soh J.E., Shimizu A., Molla M.R., Zankov D.P., Nguyen L.K., Khan M.R., Tesega W.W., Chen S., Tojo M., Ito Y., Sato A., Hitosugi M., Miyagawa S, & Ogita H. (2023). RhoA rescues cardiac senescence by regulating Parkin-mediated mitophagy. The Journal of Biological Chemistry, 299(3), 102993.
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2

Fluorescent Probes for Autophagy and Mitophagy

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Mito-Tracker Green (#M7514, MTG) and Lyso-Tracker Red (#L12492, LTR) were obtained from Invitrogen (Eugene, Oregon, USA). Autophagosome dye (#NY561, DAPGreen) and mitophagy dye (#MD01-10, Kumamoto, Japan) were obtained from Dojindo Laboratories. Carbonyl cyanide 3-chlorophenylhydrazone (#045200, CCCP) was obtained from Thermo Fisherscientific (Grand Island, NY, USA). Penicillin-streptomycin (#15140163, 10,000 units/ml), fetal bovine serum (#26140079, FBS), Dulbecco’s modified Eagle’s medium (#11965118, DMEM), and other cell culture reagents were obtained from Gibco BRL (Grand Island, NY, USA). Primary and secondary antibodies used in this study were GAPDH (#5174, Cell Signaling Technology, Beverly, MA, USA), FIP200 (#12436, Cell Signaling Technology, Beverly, MA, USA), ATG13 (#13273, Cell Signaling Technology, Beverly, MA, USA), and HRP-linked anti-rabbit IgG (#7074, Cell Signaling Technology, Beverly, MA, USA). HeLa cells were a generous gift from Dr. Carolyn M. Price’s lab (University of Cincinnati). SLC-80 cells were isolated from healthy human fibroblasts by Dr. Taosheng Huang (Cincinnati Children’s Hospital, Cincinnati, OH, USA).
Chen Q., Shao X., Hao M., Fang H., Guan R., Tian Z., Li M., Wang C., Ji L., Chao H., Guan J.L, & Diao J. (2020). Quantitative analysis of interactive behavior of mitochondria and lysosomes using structured illumination microscopy. Biomaterials, 250, 120059.
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3

Mitophagy Modulation Assay Protocol

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Galangin was purchased from Biopurify Phytochemicals Ltd (Chengdu, China), and DAPI and Mdivi-1 were purchased form Beyotime (Shanghai, China). CellTiter-Glo was purchased from Vazyme Biotech Co.,Ltd (Nanjing, China). Mitophagy dye was obtained from DOJINDO (Kumamoto, Japan).
Zhang R., Lu J., Pei G, & Huang S. (2023). Galangin Rescues Alzheimer’s Amyloid-β Induced Mitophagy and Brain Organoid Growth Impairment. International Journal of Molecular Sciences, 24(4), 3398.
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4

Quantifying Mitophagy in HLOs and sHLOs

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Mitophagy was evaluated using Mitophagy dye (#MD01, Dojindo Molecular Technology) according to the manufacturer’s protocol. Briefly, HLOs and sHLOs were rinsed with warm HCM, then treated with 100 nM of Mitophagy dye for 30 minutes at 37 °C. The cells were rinsed twice with warm HCM and incubated at 37 °C and 5% CO2. After 24 hours, cells were washed with warm HCM and stained with NucBlue™ Live ReadyProbes™ Reagent (ThermoFisher Scientific). After staining, the cells were observed on a Keyence BZ-X800 automated fluorescence microscope (Japan). The mitophagy levels were calculated using Analysis Application Hybrid cell count (Keyence) and normalized to each nucleus signal.
Kimura M., Iguchi T., Iwasawa K., Dunn A., Thompson W.L., Yoneyama Y., Chaturvedi P., Zorn A.M., Wintzinger M., Quattrocelli M., Watanabe-Chailland M., Zhu G., Fujimoto M., Kumbaji M., Kodaka A., Gindin Y., Chung C., Myers R.M., Subramanian G.M., Hwa V, & Takebe T. (2022). En Masse Organoid Phenotyping Informs Metabolic-associated Genetic Susceptibility to NASH. Cell, 185(22), 4216-4232.e16.
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5

Visualizing Mitochondrial Dynamics in HCN Cells

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HCN cells treated with miR-Con, miR-351-5p, or siMiro2 were stained with MitoTracker (Invitrogen; M7514) and LysoTracker (Thermo Fisher Scientific, Carlsbad, CA, USA; L7528) and observed under a confocal microscope. Co-localizations of MitoTracker and LysoTracker were observed by confocal fluorescence microscopy. To examine the degree of mitophagy, HCN cells transfected with miRNAs and siMiro2 were stained with a mitophagy dye (Dojindo, Japan; MD01-10) according to the manufacturer’s instructions. To induce mitophagy, HCN cells were treated with a final concentration of 10 μM CCCP. At 24 h, the HCN cells were washed twice with serum-free medium. The intensity of the mitophagy dye was measured using ZEN software (Carl Zeiss).
Woo H.N., Park S., Kim H.L., Jung M.K., Pack C.G., Park J., Cho Y., Jo D.G., Kim D.K., Mook-Jung I., Kim S.W, & Lee H. (2020). miR-351-5p/Miro2 axis contributes to hippocampal neural progenitor cell death via unbalanced mitochondrial fission. Molecular Therapy. Nucleic Acids, 23, 643-656.
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