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Rotenone

Manufactured by Bio-Techne
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Sourced in United Kingdom, United States

Rotenone is a natural compound derived from the roots of certain plants. It is commonly used as a research tool in biochemistry and cell biology laboratories to inhibit mitochondrial complex I, a key enzyme in the electron transport chain. Rotenone has been widely utilized to study cellular respiration, oxidative stress, and neurodegeneration in various experimental models.

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Lab products found in correlation

Fluoro carbonyl cyanide phenylhydrazone (fccp), by Bio-Techne (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Oligomycin a, by Bio-Techne (2 mentions) Antimycin a, by Enzo Life Sciences (2 mentions) Ptp inhibitor xxii, by Merck Group (2 mentions) Seahorse xfe24 plates, by Agilent Technologies (2 mentions) Antimycin a, by Santa Cruz Biotechnology (2 mentions) Seahorse wave software, by Agilent Technologies (2 mentions) Seahorse xfe24 machine, by Agilent Technologies (2 mentions) Forskolin, by Bio-Techne (2 mentions) Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions) Seahorse 24 well plate, by Agilent Technologies (1 mentions) Oligomycin, by Merck Group (1 mentions) Norepinephrine, by Merck Group (1 mentions) Hoechst staining, by Merck Group (1 mentions) Antimycin a, by Bio-Techne (1 mentions) Lsm 710 microscope, by Zeiss (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Poly l lysine (pll), by Merck Group (1 mentions)

15 protocols using rotenone

1

Adipocyte Bioenergetics Profiling

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Immortalized primary BA was seeded in Seahorse 24-well plates (Agilent) 40 000 cells/well in 100 μl of BA growth medium (DMEM supplemented with 10% FBS and 0.1% penicillin/streptomycin). The following day, differentiation was induced with high-induction medium (growth medium, 20 nM insulin, 1 nM triiodothyronine, 1 μM Dexamethasone, 100 μM 3-isobutyl-1-methylxanthine, 1 μM Rosiglitazone and 125 nM Indomethacin). After 2 days, the medium was replaced with BA differentiation medium (growth medium, 20 nM insulin and 1 nM triiodothyronine). The following day, the respirometry Mito-Stress assay was performed according to manufacturer instructions, following 15 min of 7α,25-OHC (1 μM) treatment where indicated. The following substances were used: Norepinephrine (1 μM, Sigma-Aldrich), oligomycin (2 μM, Sigma-Aldrich), Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 1 μM, Tocris), Rotenone (0.5 μM, Tocris), Antimycin A (0.5 μM, Tocris), Hoechst staining (10 μg/ml, Sigma-Aldrich). The number of cells labeled with Hoechst staining was calculated with Cytation 5 Cell Imaging Multi-Mode Reader for normalization.
Copperi F., Schleis I., Roumain M., Muccioli G.G., Casola S., Klingenspor M., Pfeifer A, & Gnad T. (2022). EBI2 is a negative modulator of brown adipose tissue energy expenditure in mice and human brown adipocytes. Communications Biology, 5, 280.
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2

Astrocyte Culture and Ca2+ Imaging

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Primary cortical astrocytes were cultured according to standard protocols, modified from (Schildge et al., 2013 ). Briefly, cortices of P4–7 GLAST-CreER;mGCaMP3/+;IP3R2−/−triple transgenic mouse pups were dissociated using a Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). Isolated cells were plated on a T-75 flask coated with poly-l-lysine (Sigma-Aldrich) and fed with DMEM (Life Technologies) supplemented with 10 % heat-inactivated FBS (Life Technologies) and 1 % penicillin-streptomycin (Life Technologies). After 7 days, astrocytes formed a confluent layer at the bottom of the flask. These cells were then plated on 12 mm cover glass (Thermo Fisher Scientific) coated with poly-l-lysine (Sigma-Aldrich) and Natural Mouse Laminin (Thermo Fisher Scientific) at a density of 20,000 cells per cover glass. One day after plating, expression of mGCaMP3 was induced by applying 1 μM (Z)-4-hydroxytamoxifen (H7904, Sigma-Aldrich). At least 14 days later, astrocyte Ca2+ transients were imaged using a Zeiss LSM 710 microscope, as described below. To block mPTP activity, astrocyte cultures were incubated for 1 hour prior to Ca2+ imaging in mPTP inhibitors [Cyclosporin A (inhibits cyclophilin D, Tocris, 20 μM; takes long time to dissolve and needs filtration of remaining precipitates) and Rotenone (Mitochondrial complex I inhibitor, Tocris, 10 μM)].
Agarwal A., Wu P.H., Hughes E.G., Fukaya M., Tischfield M.A., Langseth A.J., Wirtz D, & Bergles D.E. (2017). Transient opening of the mitochondrial permeability transition pore induces microdomain calcium transients in astrocyte processes. Neuron, 93(3), 587-605.e7.
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3

Inhibitor-based Mitochondrial Respiration Assay

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PTP Inhibitor XXII (CAS 765317-72-4)38 (link) was purchased from EMD Millipore. IOXI (CAS 5852-78-8) was purchased from Cayman Chemicals. Oligomycin A, FCCP (trifluorocarbonylcyanide phenylhydrazone), rotenone, and DMOG (Dimethyloxaloglycine) were purchased from Tocris Bioscience. Antimycin A was purchased from Enzo Life Sciences.
Rabbit polyclonal anti-RNF213 antibodies were generated and used as described39 (link). Other antibodies were purchased and used for immunoprecipitations and/or immunoblotting at manufacturer-recommended concentrations (Supplementary Table 5).
Banh R.S., Iorio C., Marcotte R., Xu Y., Cojocari D., Rahman A.A., Pawling J., Zhang W., Sinha A., Rose C.M., Isasa M., Zhang S., Wu R., Virtanen C., Hitomi T., Habu T., Sidhu S.S., Koizumi A., Wilkins S.E., Kislinger T., Gygi S.P., Schofield C.J., Dennis J.W., Wouters B.G, & Neel B.G. (2016). PTP1B regulates non-mitochondrial oxygen consumption via RNF213 to promote tumour survival during hypoxia. Nature cell biology, 18(7), 803-813.
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4

Mitochondrial ROS Measurement in Neutrophils

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For measurement of mitochondrial ROS (mtROS) release, 1 × 106 neutrophils were incubated with 10 μM rotenone (Tocris Bioscience, Bristol, UK), 200 μm MitoTEMPO (Cayman Chemical, Ann Arbor, MI, USA), 10 μM TX or TX plus rotenone and loaded with 2.5 μM MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 30 min. After incubation, MitoSOX™ fluorescence intensity was measured using a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) at wavelengths of 510 nm excitation and 580 nm emission.
Salinas C., Barriga K., Albornoz A., Alarcon P., Quiroga J., Uberti B., Sarmiento J., Henriquez C., Ehrenfeld P., Burgos R.A, & Moran G. (2023). Tamoxifen triggers the in vitro release of neutrophil extracellular traps in healthy horses. Frontiers in Veterinary Science, 9, 1025249.
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5

Inhibitor-based Mitochondrial Respiration Assay

Cited 1 time
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PTP Inhibitor XXII (CAS 765317-72-4)38 (link) was purchased from EMD Millipore. IOXI (CAS 5852-78-8) was purchased from Cayman Chemicals. Oligomycin A, FCCP (trifluorocarbonylcyanide phenylhydrazone), rotenone, and DMOG (Dimethyloxaloglycine) were purchased from Tocris Bioscience. Antimycin A was purchased from Enzo Life Sciences.
Rabbit polyclonal anti-RNF213 antibodies were generated and used as described39 (link). Other antibodies were purchased and used for immunoprecipitations and/or immunoblotting at manufacturer-recommended concentrations (Supplementary Table 5).
Banh R.S., Iorio C., Marcotte R., Xu Y., Cojocari D., Rahman A.A., Pawling J., Zhang W., Sinha A., Rose C.M., Isasa M., Zhang S., Wu R., Virtanen C., Hitomi T., Habu T., Sidhu S.S., Koizumi A., Wilkins S.E., Kislinger T., Gygi S.P., Schofield C.J., Dennis J.W., Wouters B.G, & Neel B.G. (2016). PTP1B regulates non-mitochondrial oxygen consumption via RNF213 to promote tumour survival during hypoxia. Nature cell biology, 18(7), 803-813.
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6

Cellular Energetics and Glycolysis Profiling

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To determine cell energetics as well as glycolysis and oxidative phosphorylation rates, cells were seeded at a density of 50,000 cells per well. Cells were counted and plated in Seahorse Xfe24 plates (Agilent) 24 h before assay and allowed to attach overnight. The assay cartridge was hydrated with Agilent equilibration buffer in a 0% CO2 chamber at 37°C overnight. One hour before assay, culture media was removed and replaced with Seahorse assay media (Seahorse basal RPMI media supplemented with 2 mM L-glutamine and 11 mM glucose for Cell Mito Stress Test). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Agilent Seahorse Xfe24 machine, according to manufacturer instructions for the Glycolysis Stress Test and Cell Mito Stress Test. FCCP and oligomycin from Cell Energy Phenotype Test kit were used with rotenone (#3616, Tocris Bioscience; Bristol, UK), 2-DG (#B1048–500, Biovision Inc.; Milpitas, CA), and antimycin A (#SC-202467A, Santa Cruz Biotechnology; Dallas, TX). Assays were analyzed using Seahorse Wave Software (Agilent). Cell density and concentration of FCCP (1 μM) were optimized according to manufacturer’s instructions.
de Wet L., Williams A., Gillard M., Kregel S., Lamperis S., Gutgesell L.C., Vellky J.E., Brown R., Conger K., Paner G.P., Wang H., Platz E.E., De Marzo A.M., Mu P., Coloff J.L., Szmulewitz R.Z, & Vander Griend D.J. (2022). SOX2 Mediates Metabolic Reprogramming of Prostate Cancer Cells. Oncogene, 41(8), 1190-1202.
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7

Cellular Energetics and Glycolysis Profiling

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To determine cell energetics as well as glycolysis and oxidative phosphorylation rates, cells were seeded at a density of 50,000 cells per well. Cells were counted and plated in Seahorse Xfe24 plates (Agilent) 24 h before assay and allowed to attach overnight. The assay cartridge was hydrated with Agilent equilibration buffer in a 0% CO2 chamber at 37°C overnight. One hour before assay, culture media was removed and replaced with Seahorse assay media (Seahorse basal RPMI media supplemented with 2 mM L-glutamine and 11 mM glucose for Cell Mito Stress Test). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Agilent Seahorse Xfe24 machine, according to manufacturer instructions for the Glycolysis Stress Test and Cell Mito Stress Test. FCCP and oligomycin from Cell Energy Phenotype Test kit were used with rotenone (#3616, Tocris Bioscience; Bristol, UK), 2-DG (#B1048–500, Biovision Inc.; Milpitas, CA), and antimycin A (#SC-202467A, Santa Cruz Biotechnology; Dallas, TX). Assays were analyzed using Seahorse Wave Software (Agilent). Cell density and concentration of FCCP (1 μM) were optimized according to manufacturer’s instructions.
de Wet L., Williams A., Gillard M., Kregel S., Lamperis S., Gutgesell L.C., Vellky J.E., Brown R., Conger K., Paner G.P., Wang H., Platz E.E., De Marzo A.M., Mu P., Coloff J.L., Szmulewitz R.Z, & Vander Griend D.J. (2022). SOX2 Mediates Metabolic Reprogramming of Prostate Cancer Cells. Oncogene, 41(8), 1190-1202.
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8

Culturing INS-1 Rat Pancreatic Cells

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The INS-1 rat pancreatic β-cell line (832/13; a gift from Dr. Christopher Newgard, Duke University, Durham, NC) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (catalog number: 11875135) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (catalog number: 10438026), 1 mM sodium pyruvate (catalog number: 11360070), 2 mM L-glutamine (catalog number: 25030081), 0.05 mM 2-mercaptoethanol (catalog number: 21985023), 100 U/mL pencillin-streptomycin (catalog number: 15140–122), all from Thermo Fisher Scientific, and 10 mM HEPES buffer (Sigma-Aldrich, catalog number: H0887). Cells were seeded in a 75 cm2 tissue culture flask in five replicates and placed at 37 0C under 5% CO2 in a humidified incubator. Cells were grown until ~80% confluent before treatment with rotenone (Catalog number: 83-79-4, Tocris, Minneapolis, MN) or thapsigargin (catalog number: 67526-95-8, Tocris) at half maximal inhibition concentration, that is, concentrations resulting in 50% cell death (IC50), which were predetermined using cell viability assays described below. All treatments were carried out for 24 h.
Weldemariam M.M., Woo J, & Zhang Q. (2022). Pancreatic INS-1 β-Cell Response to Thapsigargin and Rotenone: A Comparative Proteomics Analysis Uncovers Key Pathways of β-Cell Dysfunction. Chemical research in toxicology, 35(6), 1080-1094.
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9

Pharmacological Manipulation of Cells

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Drugs were applied by dissolving them to the desired final concentration in Krebs' solution and by switching the perfusion from control solution to drug-containing solution. Carbachol, forskolin, H-89, picrotoxin, quinpirole (Quin), rotenone (Rot), and L-sulpiride (Sulp) were purchased from Tocris-Cookson (Bristol, UK). Drugs were dissolved into the external medium or Krebs' solution and applied by switching the perfusion from control solution to drug-containing solution.
Tozzi A., Tantucci M., Marchi S., Mazzocchetti P., Morari M., Pinton P., Mancini A, & Calabresi P. (2018). Dopamine D2 receptor-mediated neuroprotection in a G2019S Lrrk2 genetic model of Parkinson’s disease. Cell Death & Disease, 9(2), 204.
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10

Melanoma and Triple Negative Breast Cancer Cell Cultures

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Vemurafenib and compound C (Dorsomorphin) were purchased from Selleck Chemicals, Houston, TX, United States; rotenone from Tocris Bioscience, Bristol, United Kingdom; and deguelin (Item №: 10010706) from Cayman Chemical, Ann Arbor, MI, United States. A375 (CRL-1619) and A2058 (CRL-11147) cell lines were purchase from ATCC, Manassas, VA, United States. Normal human melanocytes were collected from tissue donated anonymously at the University of Utah. HCC1806 triple negative breast cancer cell lysates were a gift from Dr. Siva Kolluri at Oregon State University.
Carpenter E.L., Chagani S., Nelson D., Cassidy P.B., Laws M., Ganguli-Indra G, & Indra A.K. (2019). Mitochondrial Complex I Inhibitor Deguelin induces metabolic reprogramming and sensitizes vemurafenib resistant BRAFV600E mutation bearing metastatic melanoma cells.. Molecular carcinogenesis, 58(9), 1680-1690.
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