Murashige and Skoog medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, 2-(N-morpholino)ethanesulfonic acid (MES), triphenil-tetrazolium chloride (TTC), xylenol orange, EDTA, succinate, 4-morpholinepropanesulfonic acid (MOPS), Polyvinylpyrrolidone (PVP-40), hydroxylamine, sulphanilamide, α-naphthylamine, ampicillin, NP40, safranin, Salicylhydroxamic acid (SHAM), kalium-cyanide (KCN), linoleic acid, fatty acid free bovine serum albumin (BSA), luminol, p-Coumaric acid, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), were obtained from Sigma-Aldrich. ProBond Purification System was purchased from Invitrogen. Amicon Ultra 30K Centrifugal Filter Units were purchased from Merck. IPTG was obtained from Duchefa Biochemie, cytochrome c was purchased from Fluka. Primary and secondary antibodies were purchased from Agrisera Antibodies. All other chemicals were of analytical or HPLC grade, and were purchased from Reanal, Hungary. Pierce BCA Protein Assay Kit, GeneJET Plant RNA Purification Kit, and RevertAid First-Strand cDNA Synthesis Kit were obtained from Thermo Scientific; SensiFAST SYBR No-ROX Kit was purchased from Bioline.
Carbonyl cyanide p trifluoromethoxyphenylhydrazone
Manufactured by Merck Group
Sourced in United States
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone is a chemical compound used in laboratory settings. It functions as an uncoupler of oxidative phosphorylation in mitochondria.
Automatically generated - may contain errors
Lab products found in correlation
Oligomycin, by Merck Group (6 mentions)
Rotenone, by Merck Group (6 mentions)
Antimycin a, by Merck Group (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Xfp extracellular flux analyzer, by Agilent Technologies (2 mentions)
Xfp microplates, by Agilent Technologies (2 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Sensifast sybr no rox kit, by Meridian Bioscience (1 mentions)
Luminol, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Np 40, by Merck Group (1 mentions)
Polyvinylpyrrolidone, by Merck Group (1 mentions)
Hydroxylamine, by Merck Group (1 mentions)
Probond purification system, by Thermo Fisher Scientific (1 mentions)
Safranin, by Merck Group (1 mentions)
Sulphanilamide, by Merck Group (1 mentions)
Genejet plant rna purification kit, by Thermo Fisher Scientific (1 mentions)
13 protocols using carbonyl cyanide p trifluoromethoxyphenylhydrazone
1
Plant Cell Culture Optimization
2
Neuronal Metabolic Profiling with XFp
3
Evaluating Mitochondrial Dysfunction in Cells
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Ciclopirox olamine (CPX) was purchased from Dibo Chemical Technology Limited Company (Shanghai, China). BTZ was obtained from Targetmol (MA, USA). Antimycin A, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, oligomycin, and rotenone were purchased from Sigma Aldrich (MO, USA). The Cell Mitochondria Isolation Kit, crystal violet, MTT cell proliferation and cytotoxicity kits, N-acetyl cysteine (NAC), and Nuclear and Cytoplasmic Protein Extraction Kits were purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kits were purchased from BD Biosciences (CA, USA), Pierce BCA Protein Assay Kits and TRIzol reagent from Thermo Fisher Scientific (MA, USA), and Protease (cOmplete Mini) and phosphatase-inhibitor cocktail tablets (PhosSTOP) from Roche Applied Science (Penzberg, Germany).
4
Metabolic Regulation in Cell Lines
5
Measuring Cellular Respiration in WT and Mutant Cells
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OCR in WT and G3842A mutant cells were determined using an XF24 extracellular flux analyzer (Seahorse Bioscience, Santa Clara, CA, USA), as previously described [25 (link)]. After calibration, the basic respiration, ATP production (using 1 μM oligomycin; BBI Life Science, Shanghai, China), and maximum respiratory capacity (using 0.5 μM carbonyl cyanide-p-trifluoromethoxy phenylhydrazone; Sigma-Aldrich) were measured.
6
Bacterial Persister Induction and Antibiotic Treatments
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Various Gram-negative (E. coli, P. aeruginosa, Acinetobacter baumannii, Klebsiella Pneumoniae, Shigella flexneri, Salmonella typhimurium, and Aeromonas hydrophila) and Gram-positive (S. aureus, Bacillus subtilis, Bacillus thuringiensis, and Staphylococcus epidermidis) bacterial strains were used in this study and their characteristics are described in Supplementary Table S1 . For normal cell culturing, three mediums were used: M9 medium plus 5 g/L glucose, Luria-Bertani (LB) medium, or Mueller-Hinton broth. M9 medium with and without 2 g/L fumarate were used for nutrient shift- and starvation-induced E. coli persister formation, respectively. Yeast nitrogen broth medium was used for starvation-induced persister formation in S. aureus. Antibiotics used in this study include tobramycin, streptomycin, gentamicin, kanamycin, ampicillin, ofloxacin, with their manufacturers and final concentrations for different treatments being described in Supplementary Table S2 . Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and its analog FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) were purchased from Sigma-Aldrich. All other chemical reagents are of analytical purity.
7
Mitochondrial Membrane Potential Assay using JC-1
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The fluorescent dye JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine Iodide, AdipoGen) was used to assess the mitochondrial membrane potential. JC-1 is a lipophilic cation dye that exhibits a potential-dependent accumulation in mitochondria indicated by a fluorescence emission shift from green (529 nm) to red (590 nm) [50 (link)]. Cells were seeded in a Petri dish and treated with 200 µM of H2O2 for 1 h; subsequently, hydrogen peroxide was removed, cells were washed once with PBS and then incubated with 5 μg/mL JC-1, at 37 °C for 15 min. After two washes with PBS, cells were covered with fresh PBS and their fluorescence was analyzed using a Zeiss Axiophot (Carl Zeiss) fluorescent microscope. Mitochondrial uncoupler FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, Sigma) was used as a positive control.
Acquired images were analyzed with ImageJ (NIH, Bethesda, MD, USA) for green and red fluorescence and results were given as red/green fluorescence ratio. At least 100 cells per sample were analyzed.
Acquired images were analyzed with ImageJ (NIH, Bethesda, MD, USA) for green and red fluorescence and results were given as red/green fluorescence ratio. At least 100 cells per sample were analyzed.
8
Mitochondrial Respiration Analysis in Adipocytes
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Oxygen consumption rate (OCR), ECAR, and proton production rate (PPR) were measured at 37°C using a XF-96 extracellular flux analyzer (Agilent Technologies). At day 7 of differentiation, the medium was replaced with prewarmed unbuffered DMEM (5.5 mM glucose) supplemented with 2, 3, or 4% essentially fatty acid–free BSA and incubated at 37°C in a non–carbon dioxide (CO2) incubator for 1 h. Basal respiration was measured in untreated cells. Coupled respiration was inhibited by oligomycin treatment (5 μM). UCP1-mediated uncoupled respiration was determined after isoproterenol (0.5 μM) stimulation. Maximum respiratory capacity was assessed after carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (Sigma-Aldrich) stimulation (7 μM). Finally, mitochondrial respiration was blocked by antimycin A (Sigma-Aldrich) (5 μM), leaving only non-mitochondrial OCR to be measured. OCR, ECAR, and PPR were automatically calculated by the Seahorse XF-96 software.
9
Mitochondrial Respiration Measurement
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Mitochondrial respiratory activity was determined by measuring cellular O 2 consumption using high-resolution respirometry with the Oxygraph O2K system (Oroboros, Innsbruck, Austria) based on a Clark-electrode, as described previously (16) . The measurement chambers were filled with Roswell Park Memorial Institute (RPMI) medium (Sigma-Aldrich, St. Louis, Mo), maintained at 37°C and allowed to equilibrate O 2 concentration with the surrounding air. 10 × 10 6 intact cells were added to each measurement chamber, and routine respiration (basal O 2 consumption rate) was recorded. Leak respiration independent of ATP production was determined by adding oligomycin (2.5 μM, Sigma-Aldrich, St. Louis, Mo). The stepwise addition of the uncoupler FCCP (Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone, 0.25 μM, Sigma-Aldrich) until no further increase in O 2 consumption was detectable reflects the maximum respiration or electron transport system (ETS) capacity. Rotenone (0.5 μM, Sigma-Aldrich) inhibits complex I, while antimycine (2.5 μM, Sigma-Aldrich) inhibits complex III, which allowed measuring residual O 2 consumption.
10
Mitochondrial Stress Response Profiling
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Mitochondrial stress tests were performed to measure oxygen consumption using the Seahorse Xfe96 Analyzer (Agilent) according to the manufacturer’s protocol. Cultivated cells were transferred to a poly-D-lysine–coated (Sigma), 96-well seahorse plate and incubated for at least 1 hour in a CO2-free incubator in XF Seahorse medium (XF Dulbecco's Modified Eagle Medium, 10mM glucose, 1mM pyruvate, and 2mM glutamine) (all Agilent) at 37°C. A Seahorse Xfe96 Analyzer was used to inject 10 μM oligomycin (Sigma), 15 μM Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (Sigma), and rotenone 1 μM (Sigma) + antimycin A 10 μM (Sigma). The Seahorse Xfe96 Analyzer was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after each injection.
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