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Mitob

Manufactured by Cayman Chemical
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MitoB is a fluorescent probe that can be used to detect and quantify mitochondrial superoxide in living cells. It is a cell-permeable molecule that selectively accumulates in the mitochondria, where it can be oxidized by superoxide to produce a fluorescent product.

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Lab products found in correlation

Olaparib, by Cayman Chemical (1 mentions) Diamide, by Merck Group (1 mentions) Ferrostatin, by Cayman Chemical (1 mentions) Dehydroascorbic acid, by Merck Group (1 mentions) Sodium ascorbate, by Merck Group (1 mentions) Liproxstatin, by Cayman Chemical (1 mentions) Auranofin, by Cayman Chemical (1 mentions) Ascorbic acid, by Merck Group (1 mentions) Lcms 8060 triple quadrupole mass spectrometer, by Shimadzu (1 mentions) Nexera x2 uhplc, by Shimadzu (1 mentions)

4 protocols using mitob

1

Synthesis and Preparation of Mitochondrial Probes

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The probe compounds MitoB, MitoP, and their deuterium spikes deuterium15-MitoB (d15MitoB) and deuterium15-MitoP (d15MitoP) were synthesized at the University of Glasgow according to Cairns, et al.45 and kept as a crystalline solid at −20 °C. MitoB is already commercially available (Cayman Chemical, Ann Arbor, Michigan, USA), and commercial development of the other 3 compounds is ongoing, but in the meantime RCH can provide the four compounds to interested researchers on request. If large quantities are required, there is also a detailed published protocol for the synthesis of these compounds45 . A MitoB solution was produced by dissolving MitoB powder in absolute ethanol heated to 60 °C; this was then diluted to 504 μM MitoB in a sterile saline solution (0.9% (w/v) NaCl/H2O), with a final ethanol content of 1% (v/v). A 504 μM MitoP solution was produced in the same way. Aliquots of MitoB, MitoP and 0.9% saline solutions were stored at −70 °C and thawed at the time of the experiment. Standard solutions for the calibration curve and for the deuterium spikes were made by serially diluting the compounds in 100% ethanol; these were also kept at −70 °C.
Salin K., Auer S.K., Villasevil E.M., Anderson G.J., Cairns A.G., Mullen W., Hartley R.C, & Metcalfe N.B. (2017). Using the MitoB method to assess levels of reactive oxygen species in ecological studies of oxidative stress. Scientific Reports, 7, 41228.
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2

Investigating Redox Signaling Pathways

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Sodium ascorbate (Sigma A4034)
Ascorbic acid (Sigma A5960)
DehydroAscorbic acid (Sigma 261556)
H2O2 (VWR BDH7103)
Olaparib (Cayman 10621)
Diamide (Sigma 87751)
Deferoxamine mesylate (DFO; Cayman 14595)
Imidazole ketone erastin (IKE; Cayman 27088)
FIN56 (Cayman 25180)
RSL3 (Cayman 19288)
Auranofin (Cayman 15316)
Ferrostatin (Cayman 17729)
Liproxstatin (Cayman 17730)
Seahorse PMP Reagent (Agilent 102504-100)
MitoB (Cayman 17116)
MitoP (Cayman 17117)
MitoB-d15 (Cayman 17470)
MitoP-d15 (Cayman 19296)
Jankowski C.S, & Rabinowitz J.D. (2022). Selenium modulates cancer cell response to pharmacologic ascorbate. Cancer research, 82(19), 3486-3498.
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3

In Vivo Mitochondrial H2O2 Quantification

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Measurement of in vivo mitochondrial H2O2 production was performed according to the previously published protocol with modifications to the LC-MS/MS method(Cocheme et al., 2012 (link)). In brief, mice were injected retro-orbitally with 0.8 μmol/kg MitoB (Cayman Chemical, cat# 17116). Mice were euthanized 3 hours after injection and 100 mg of tissues were clamp-frozen. Tissues were processed as previously described previously (Cocheme et al., 2012 (link)). MitoB and MitoP compounds were analyzed by injecting 1 μL of sample into the LC/MS/MS system consisting of a Shimadzu LCMS-8060 triple quadrupole mass spectrometer operating the DUIS ion source in electrospray positive mode coupled to a Nexera X2 UHPLC (Shimadzu Scientific Instruments, Columbia, MD). Compounds were resolved on a Fluorophenyl column (2.7 micron, 2.1×100 mm, Restek Corporation, Bellefonte, PA) maintained at 30°C. The mobile phase consisted of water containing 0.1% formic acid (eluent A) and acetonitrile containing 0.1% FA (eluent B). Gradient elution was performed starting at 20% eluent B, a linear increase to 75% eluent B until 2.0 minutes, a step to 100% eluent B until 2.1 minutes, 100% eluent B until 5.6 minutes and re-equilibration from 5.7 until 7 minutes. The flow rate was set to 0.5 mL/minute.
Crewe C., Funcke J.B., Li S., Joffin N., Gliniak C.M., Ghaben A.L., An Y.A., Sadek H.A., Gordillo R., Akgul Y., Chen S., Samovski D., Fischer-Posovszky P., Kusminski C.M., Klein S, & Scherer P.E. (2021). Extracellular vesicle-Based Interorgan Transport of Mitochondria from Energetically Stressed Adipocytes. Cell metabolism, 33(9), 1853-1868.e11.
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4

Hepatocyte Culture with Isotope Labeling

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Three hours after seeding the hepatocytes, the culture medium was changed to the new basal medium containing 5-μM mitoB (Cayman Chemical, 17116). To replace oxygen with 18O2 as much as possible, a bubbling treatment was carried out with 18O2-containing gas in the new basal medium [Gas A (N2: 75%, 18O2: 20%, CO2: 5%) for 37 °C culture and Gas B (N2: 80%, 18O2: 20%) for 4 °C culture]. Gas mixing was completed by a custom-made gas mixer. After the medium change, the cell culture plates were put into a chamber that had been incubated at 37 °C in advance. Then, the chamber was filled with 18O2-containing gas for 30 min at a flow rate of 20 cc/min, which was presumed to replace the gas in the chamber completely. After that, each chamber was placed in a 37 °C incubator or a 4 °C refrigerator and cultured for 12 h. Then, the cells were washed with PBS once and homogenized in 1-mL MeOH (5 × 105 cells/mL). These homogenates were stored at −80 °C and used for measurements of mitoP/B39 (link),40 (link) and lipids within 2 weeks.
Anegawa D., Sugiura Y., Matsuoka Y., Sone M., Shichiri M., Otsuka R., Ishida N., Yamada K.I., Suematsu M., Miura M, & Yamaguchi Y. (2021). Hepatic resistance to cold ferroptosis in a mammalian hibernator Syrian hamster depends on effective storage of diet-derived α-tocopherol. Communications Biology, 4, 796.
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