Rotenone and antimycin a

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Rotenone and antimycin A are chemical compounds commonly used as research tools in life science laboratories. Rotenone is a complex organic molecule derived from certain plant species, while antimycin A is a macrolide antibiotic. Both compounds are frequently employed as inhibitors of the electron transport chain in mitochondria, a key cellular process involved in energy production. The core function of these compounds is to disrupt cellular respiration, making them valuable for studying various biological and biochemical mechanisms. However, a more detailed description cannot be provided while maintaining an unbiased and strictly factual approach, as that would require interpretation or extrapolation beyond the core functionality.

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Lab products found in correlation

Close spelling variants of this product

Rotenone (81 citations) Antimycin A (73 citations) Rotenone/antimycin A (66 citations) Antimycin (11 citations) Rotenone/antimycin (4 citations) Rotenone and antimycin (2 citations)

5 protocols using rotenone and antimycin a

Investigation of Redox Signal Origins

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To investigate the origin of redox signals, various inhibitors such as sodium diethyldithiocarbamate, GKT136901 (Sigma‐Aldrich, MO, USA), Mito‐tempo (MedChemExpress, NJ, USA), FCCP, oligomycin, rotenone and antimycin A (Agilent, CA, USA), and Batimastat (Selleckchem, Seoul, South Korea) were used as inhibitors. All of the inhibitors were incubated for 24 h at 37 °C. Furthermore, the cells were treated with CPI‐613 (MedChemExpress, NJ, USA) for drug screening and incubated for 24 h at 37 °C. After treatment, the cells were washed with DPBS for EC detection within 30 s and cytotoxicity testing.

Koo K., Kim C., Kim H., Cho Y., Suhito I.R, & Kim T. (2023). Extracellularly Detectable Electrochemical Signals of Living Cells Originate from Metabolic Reactions. Advanced Science, 10(9), 2207084.

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Seahorse XFe96 Analyzer-based Mitochondrial Respiration

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HuH-7 cells and their derivatives were plated in a Seahorse XF96 Cell Culture Microplate (Agilent Technologies, Santa Clara, CA, USA) at a density of 12,000 cells in 80 μL DMEM supplemented with 5% FBS and antibiotics per well and were incubated overnight at 37 °C in 5% CO₂. The culture medium was replaced with XF DMEM (Agilent) supplemented with glucose (10 mM), L-glutamine (2 mM), and sodium pyruvate (1 mM) at pH 7.4, and cells were further incubated at 37 °C in a non-CO₂ incubator for 1 h. The OCR and the extracellular acidification rate (ECAR) were measured using the Agilent Seahorse XFe96 Analyzer and mitochondrial reparation was assessed by sequential addition of 1.5 μM oligomycin (Agilent), 0.5 μM fluoro-carbonyl cyanide phenylhydrazone (Agilent), and 1.0 μM rotenone and antimycin A (Agilent), according to the manufacturer’s method. After analysis, cells were fixed in 70% ethanol and stained with Hoechst 33,258 to count the remaining cells. Cell numbers were calculated from fluorescent images processed with Image-J software and used to normalize OCR and ECAR data.

Sasaki M., Yamamoto K., Ueda T., Irokawa H., Takeda K., Sekine R., Itoh F., Tanaka Y., Kuge S, & Shibata N. (2023). One-carbon metabolizing enzyme ALDH1L1 influences mitochondrial metabolism through 5-aminoimidazole-4-carboxamide ribonucleotide accumulation and serine depletion, contributing to tumor suppression. Scientific Reports, 13, 13486.

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Bioenergetic Profiling of Drug Effects

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The bioenergetic function of cells in response to drug treatments was determined using a Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Cells were seeded in specialized V7 Seahorse tissue culture plates (Agilent, 102601-100) for 24 hours. Cells were then treated with the indicated concentrations of BAS-2 for further 24 hours. One hour before the experiment, cells were washed and changed to XF Base medium (Agilent, 10353-100) adjusted to pH 7.4. For oxidative phosphorylation experiments, medium was supplemented with pyruvate (1 mM), l-glutamine (2 mM), and glucose (10 mM). Three baseline OCR measurements were taken, followed by three measurements after each injection of oligomycin (1 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 0.5 μM), and rotenone and antimycin A (0.5 mM) (Agilent, 103015-100), respectively. For glycolysis experiments, medium was supplemented with l-glutamine (1 mM). Three baseline measurements were taken for ECAR, followed by three measurements after each injection of glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM) (Agilent, 103020-100), respectively. For all analysis, values were normalized to protein concentration before baseline measurements were subtracted. The fold change was calculated relative to DMSO.

Dowling C.M., Hollinshead K.E., Di Grande A., Pritchard J., Zhang H., Dillon E.T., Haley K., Papadopoulos E., Mehta A.K., Bleach R., Lindner A.U., Mooney B., Düssmann H., O’Connor D., Prehn J.H., Wynne K., Hemann M., Bradner J.E., Kimmelman A.C., Guerriero J.L., Cagney G., Wong K.K., Letai A.G, & Chonghaile T.N. (2021). Multiple screening approaches reveal HDAC6 as a novel regulator of glycolytic metabolism in triple-negative breast cancer. Science Advances, 7(3), eabc4897.

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Metabolic Profiling of Bone Marrow-Derived Macrophages

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One day prior to the assay, BMDM were plated at 5 x 10⁴ cells/well in a 96-well Seahorse assay plate. All measurements were performed on a Seahorse XF^e96 Extracellular Flux Analyzer (Agilent). The glycolysis and mitochondrial stress tests were performed using the manufacturer’s protocol. Briefly, extracellular acidification rate was measured at baseline and after the addition of 10 mM glucose (Sigma-Aldrich), 1 mM oligomycin (Agilent), and 50 mM 2-deoxyglucose (2-DG; Sigma-Aldrich). Oxygen consumption rate was measured at baseline and after the addition of 1 mM oligomycin, 1 mM FCCP (Agilent), and 0.5 mM antimycin A and rotenone (Agilent).

Stothers C.L., Burelbach K.R., Owen A.M., Patil N.K., McBride M.A., Bohannon J.K., Luan L., Hernandez A., Patil T.K., Williams D.L, & Sherwood E.R. (2021). Beta-glucan Induces Distinct and Protective Innate Immune Memory in Differentiated Macrophages. Journal of immunology (Baltimore, Md. : 1950), 207(11), 2785-2798.

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Metabolic Phenotyping of Cells

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Cells were plated in a 96-well Seahorse assay plate at 4×10⁴ cells/well in Seahorse Assay Media and assessed on the Seahorse XF^e96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA). For the glycolysis stress test, cells were sequentially treated with 10mM glucose (RPI, Mount Prospect, IL), 1μM oligomycin (Agilent Technologies), and 50mM 2-deoxyglucose (Sigma-Aldrich). For the mitochondrial stress test, Assay media was supplemented with 10mM glucose and cells were sequentially treated with 1μM oligomycin (Agilent Technologies), 1μM FCCP (Agilent Technologies), and 0.5 μM of antimycin A and rotenone (Agilent Technologies).

Fensterheim B.A., Young J.D., Luan L., Kleinbard R.R., Stothers C.L., Patil N.K., McAtee-Pereira A.G., Guo Y., Trenary I., Hernandez A., Fults J.B., Williams D.L., Sherwood E.R, & Bohannon J.K. (2018). The TLR4 agonist monophosphoryl lipid A drives broad resistance to infection via dynamic reprogramming of macrophage metabolism. Journal of immunology (Baltimore, Md. : 1950), 200(11), 3777-3789.

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