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Carbonyl cyanide 4

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Carbonyl cyanide 4 is a laboratory reagent used for specific purposes in scientific research and analysis. It is a colorless, volatile liquid with a distinctive odor. The core function of this compound is to serve as a chemical tool for targeted applications in controlled laboratory environments.

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Lab products found in correlation

Antimycin a, by Merck Group (7 mentions) Rotenone, by Merck Group (6 mentions) Oligomycin, by Merck Group (4 mentions) Phenylhydrazone, by Merck Group (3 mentions) Sodium pyruvate, by Merck Group (2 mentions) Oligomycin a, by Merck Group (2 mentions) Cell tak, by Corning (2 mentions) D glucose, by Merck Group (2 mentions) Inh h151, by InvivoGen (2 mentions) Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (2 mentions) Ru360, by Merck Group (2 mentions) 2 deoxyglucose, by Merck Group (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Sodium palmitate, by Merck Group (1 mentions) Oxamate, by Merck Group (1 mentions) Myxothiazol, by Merck Group (1 mentions) Aminooxyacetate, by Merck Group (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Mitotracker green fm, by Thermo Fisher Scientific (1 mentions) 2 deoxy d glucose, by Merck Group (1 mentions)

Spelling variants of this product

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (118 citations) Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (26 citations) Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazon (FCCP, (2 citations)

12 protocols using carbonyl cyanide 4

1

Mitochondrial Respiration Assay Compounds

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Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), myxothiazol, antimycin A, rotenone, 2-deoxyglucose, oxamate, aminooxyacetate, glucose, sodium pyruvate and sodium palmitate were obtained from Sigma (St. Louis, MO, USA). L-glutamine was obtained from Invitrogen (Carlsbad, CA). Oligomycin was obtained from EMD (San Diego, CA, USA). Bovine Serum Albumin fraction V (fatty acid ultra-free) was obtained from Roche Diagnostics (Indianapolis, IN, USA). All compounds and medium were prepared according to the manufacturers' instructions unless indicated otherwise.
Pike Winer L.S, & Wu M. (2014). Rapid Analysis of Glycolytic and Oxidative Substrate Flux of Cancer Cells in a Microplate. PLoS ONE, 9(10), e109916.
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2

Mitochondrial Metabolism Profiling Reagents

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Protease inhibitor and Phosphate inhibitor cocktails, 2-Deoxy-D-glucose, Oligomycin A, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, Antimycin A and Rotenone were purchased from Sigma-Aldrich. HTH-01-015, a potent and selective NUAK1 inhibitor (23 (link)) was from Tocris (Bristol, UK). Fluorophores Tetramethylrhodamine Ethyl Ester, Perchlorate (TMRE), MitoTracker™ Green FM, and Hoechst 33,342 were from ThermoFisher. AccuRuler RGB plus protein ladder was purchased from MaestroGen Inc. (Hsinchu City, Taiwan). Anti-NUAK1 antibody (#4458) was from Cell Signaling (Danvers, MA, USA), and the anti-FLAG (M2) was from Sigma-Aldrich. Antibodies against β-Actin (AC-15), ATP5B (E-1), and TOM20 (F-10) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Total OXPHOS Rodent WB Antibody Cocktail (ab110413) was from Abcam (Cambridge, United Kingdom). Goat Secondary antibodies anti-mouse IgG-HRP and anti-rabbit IgG-HRP conjugates were purchased from Bio-Rad (Hercules, CA, USA). The anti-mouse Alexa-488 antibody (A11001) was from ThermoFisher.
Escalona E., Muñoz M., Pincheira R., Elorza Á.A, & Castro A.F. (2020). Cytosolic NUAK1 Enhances ATP Production by Maintaining Proper Glycolysis and Mitochondrial Function in Cancer Cells. Frontiers in Oncology, 10, 1123.
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3

CD4+ T Cell Metabolic Profiling

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In vitro stimulated human (anti-CD3 + anti-CD46) or mouse (anti-CD3 + anti-CD28) CD4+ T cells (stimulated for 22–24 hours) were resuspended in serum-free unbuffered Seahorse XF RPMI medium (#103576–100, Agilent Technologies, Santa Clara,CA) that was supplemented with glucose, glutamine and sodium pyruvate. These CD4 T cells were plated onto Cell-Tak (#354241; Corning, Reinach, Switzerland) coated seahorse cell plates at 2.5×105 per well. Metabolic profiling was achieved by the perturbation of specific metabolic pathways by the addition of oligomycin (#O4876; 1 μM), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, #C2920; 2 μM) and rotenone (#R8875; 1 μM) +/− Antimycin A (#A8674; 0.5uM) - all from Sigma Aldrich, St. Louis, MO). Metabolic parameters were then calculated based on the following formulas:
West E.E., Merle N.S., Kamiński M.M., Palacios G., Kumar D., Wang L., Bibby J.A., Overdahl K., Jarmusch A.K., Freeley S., Lee D.Y., Thompson J.W., Yu Z.X., Taylor N., Sitbon M., Green D.R., Bohrer A., Mayer-Barber K.D., Afzali B., Kazemian M., Scholl-Buergi S., Karall D., Huemer M, & Kemper C. (2023). Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection. Immunity, 56(9), 2036-2053.e12.
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4

Mitochondrial Respiration Analysis in C2C12 Cells

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Assessment of mitochondrial respiration in C2C12 skeletal muscle cells was performed using an XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA) as described by Gerencser et. al., with modifications [34 (link)]. Briefly, cells were seeded into 20 wells of a XF24 V7 microplate at a density of 5,000 cells per well. Cells were grown to 80% confluence and differentiated into myotubes. Experiments were conducted in serum free media containing 25mmol glucose, 110mg/L pyruvate, and 4mM glutamate. Experiments consisted of 3-minute mixing, 2-minute wait, and 3-minute measurement cycles. Oxygen consumption was measured as previously described under basal conditions, in the presence of the ATP synthase inhibitor, oligomycin (state 4O; 0.5 μM, O4876; Sigma-Aldrich, St. Louis, MO), and in the presence of Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (state 3u; FCCP; 300 μM, C2920; Sigma-Aldrich, St. Louis, MO) a mitochondrial uncoupler, to assess maximal respiration [34 (link),35 (link)]. Cellular respiratory control ratio (cRCR) was calculated as the ratio of FCCP stimulated maximal respiratory rate over oligomycin induced state 4 respiration. Data are expressed as percent change from baseline values to account for potential differences in cell number across wells. All experiments were performed at 37 °C.
Frisard M.I., Wu Y., McMillan R.P., Voelker K.A., Wahlberg K.A., Anderson A.S., Boutagy N., Resendes K., Ravussin E, & Hulver M.W. (2014). Low levels of lipopolysaccharide modulate mitochondrial oxygen consumption in skeletal muscle. Metabolism: clinical and experimental, 64(3), 416-427.
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5

Metabolomics Analyses Protocols

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Reagents used for metabolomics analyses included: methanol and ultra-pure water (LC-MS grade, EMD Millipore, Gibbstown, NJ); formic acid, acetic acid (Optima LC/MS grade; Fisher Chemical, Pittsburgh, PA); and butylated hydroxytoluene (BHT, TCI America; Portland, OR), as well as zirconium oxide beads (Next Advance; Averill Park, NY). Deuterium (d) labeled internal standards DHA-d5, ARA-d8, EPA-d5, LA-d4, and 9(S)-HODE-d4 (Cayman Chemical, Ann Arbor, MI) were used for quantification of total and free fatty acids, and oxidized DHA derivatives, respectively. Sterile d6-α-tocopherol emulsion (Fresenius-Kabi, Graz, Austria) and D-(+)-glucose (Sigma Aldrich, St. Louis MO) were used for embryo microinjection rescue studies. Reagents used for bioenergetics profiling included: oligomycin (Cayman Chemicals; Ann Arbor, MI), carbonyl cyanide 4-(trifluoromethoxy) phenyl-hydrazone (FCCP), and sodium azide (Sigma-Aldrich; St. Louis, MO).
McDougall M., Choi J., Kim H.K., Bobe G., Stevens J.F., Cadenas E., Tanguay R, & Traber M.G. (2017). Lethal Dysregulation of Energy Metabolism During Embryonic Vitamin E Deficiency. Free radical biology & medicine, 104, 324-332.
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6

Mitochondrial Respiration and ATP Levels

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The oxygen consumption rate (OCR) was measured using a Seahorse XFe96 Analyzer (Agilent, Santa Clara, CA, USA). SH-SY5Y cells were grown in Seahorse XF96 microplates (20,000 cells per well) for 72 h post knock-down. SH-SY5Y cells OCR analysis was performed in sodium bicarbonate and phenol red free DMEM (#D5030, Sigma-Aldrich) supplemented with l-glutamine 4 mM, d-glucose 25 mM, pH = 7.4, at 37 °C without CO2. Baseline OCR was measured followed by sequential injection of the following drugs: oligomycin 1 μM (#75351, Sigma Aldrich), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone 1.5 μM (FCCP, # C2920 Sigma Aldrich), and Antimycin A 0.5 μM (# A8674 Sigma Aldrich) with rotenone 0.5 μM (#R8875, Sigma Aldrich). OCR values were reported according to the Seahorse XF Cell Mito Stress Test (Agilent) and values obtained in pmolO2/min were normalized to the total amount of protein. Representative traces are shown in Figure 1D with each point representing the average values of 6 different independent cultures.
Total ATP levels were measured using the CellTiter-Glo® Luminescent Cell Viability Assay (# G7570, Promega, Madison, WI, USA) according to the manufacturer’s instructions. In the negative control condition, cells were pre-treated for 1 h with 5 µM mitochondrial complex III inhibitor Antimycin A. Luminescence was normalized to protein concentration.
Dentoni G., Naia L, & Ankarcrona M. (2022). Mitochondria-Endoplasmic Reticulum Interplay Regulates Exo-Cytosis in Human Neuroblastoma Cells. Cells, 11(3), 514.
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7

Calcification Modulation in RH30 Cells

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RH30 cells are cultured in 24-well tissue culture plate at the concentration of 200,000 cells per well and allowed to adhere overnight (o/n) before o/n treatment with complete growth medium supplemented with following concentrations of CaCl2 and Na3PO4: Very Low CaP: 0.7575 mM CaCl2 and 5.63 mM Na3PO4, Low CaP: 1.095 mM CaCl2 and 6.13 mM Na3PO4; Moderate CaP: 1.77 mM CaCl2 and 6.63 mM Na3PO4 for different time points. Different reagents used in calcification experiments include Ru360 (557,440, Sigma-Aldrich), Antimycin A (A8674, Sigma-Aldrich), Carbonyl cyanide 4-(trifluoromethoxy) phenyl-hydrazone Ready Made Solution, FCCP (SML2959, Sigma-Aldrich), Universal Type I IFN Protein (Recombinant Human IFN-alpha A/D [BglII]), 11,200–1, R&D systems), H-151 (inh-h151, InvivoGen), and VBIT-4 (S3544, Selleckchem).
Duvvuri B., Pachman L.M., Hermanson P., Wang T., Moore R., Wang D.D., Long A., Morgan G.A., Doty S., Tian R., Sancak Y, & Lood C. (2023). Role of mitochondria in the myopathy of juvenile dermatomyositis and implications for skeletal muscle calcinosis. Journal of autoimmunity, 138, 103061.
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8

Measuring Mitochondrial Respiration in BMDMs

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The oxygen consumption rate (OCR) was measured using a Seahorse XF-96 Flux Analyzer (Seahorse Biosciences, Billerica, MA). The culture medium consisted of DMEM supplemented with 10% FBS and penicillin-streptomycin-amphotericin B. BMDMs were seeded in an XF-96 culture plate at a density of 1 × 105 cells/well and incubated overnight. The cells were treated with FAC for 16 h. The assay medium comprised the XF base medium (Seahorse Biosciences) supplemented with 5.5 mM D-glucose (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), and 1X GlutaMAX™ (Gibco), adjusted to pH 7.4. The inhibitors were used in the following concentrations: oligomycin A (2 μmol/L; Sigma-Aldrich), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP, 1 μmol/L; Sigma-Aldrich), rotenone (3 μmol/L; Sigma-Aldrich), and antimycin A (3 μmol/L; Sigma-Aldrich). In short, oligomycin inhibits ATP synthase (complex V), and the decrease in OCR represents cellular ATP production. FCCP is an uncoupling agent to calculate spare respiratory capacity. A combination of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor, shuts down mitochondrial respiration and enables the calculation of nonmitochondrial respiration.
Choi E.J., Jeon C.H, & Lee I.K. (2022). Ferric Ammonium Citrate Upregulates PD-L1 Expression through Generation of Reactive Oxygen Species. Journal of Immunology Research, 2022, 6284124.
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9

Mitochondrial Membrane Potential Assay

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Fluorescent cationic probe tetramethylrhodamine methyl ester (TMRM) (Sigma-Aldrich, St. Louis, USA) was used to evaluate the ΔΨ. As a control, we used the uncoupling agent Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma-Aldrich, St. Louis, USA). Cells were plated in 96-well plates at a density of 60,000/well for neurons or 15,000/well for astrocytes. Cells were incubated for 30 min at 37°C with TMRM (30 nM) with or without FCCP (10 μM) diluted in KREBS medium. Cells were then washed with KREBS solution and fluorescence was read with the plate reader VICTOR® Multilabel Plate Reader (PerkinElmer). Data were normalized to the total amount of protein measured by the Bradford assay kit (Bio-Rad Laboratories, California, USA).
Contino S., Suelves N., Vrancx C., Vadukul D.M., Payen V.L., Stanga S., Bertrand L, & Kienlen-Campard P. (2021). Presenilin-Deficient Neurons and Astrocytes Display Normal Mitochondrial Phenotypes. Frontiers in Neuroscience, 14, 586108.
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10

Evaluating Cellular Stress in Insect Cells

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Fetal bovine serum (FBS) was purchased from LGC Biotecnologia (Cotia, SP, Brazil). The Schneider’s Insect Medium (SIM), Dulbecco’s Modified Eagle Medium (DMEM), and Grace’s Insect Medium (GIM), amphotericin B solution (AmB, 250 µg/mL), penicillin-streptomycin solution (5000 units penicillin and 5 mg streptomycin/mL), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), thiazolyl blue tetrazolium Bromide (MTT), 2,2′ azobis (2-methylpropionamidine) dihydrochloride (AAPH), 2′,7′-dichlorofluorescin diacetate (H2DCFDA), lipopolysaccharide (LPS), and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was purchased from Synth (Diadema, SP, Brazil). A Panoptic staining kit was obtained from Laborclin (Pinhais, PR, Brazil). All other reagents were analytical grade.
Garcia A.R., Amorim M.M., Amaral A.C., da Cruz J.D., Vermelho A.B., Nico D, & Rodrigues I.A. (2023). Anti-Leishmania amazonensis Activity, Cytotoxic Features, and Chemical Profile of Allium sativum (Garlic) Essential Oil. Tropical Medicine and Infectious Disease, 8(7), 375.
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