RPMI 1640, penicillin, streptomycin and PBS were purchased from Gibco, Lifetechnologies (Darmstadt, Germany). FCS was purchased from Biochrom (Berlin, Germany). RIPA buffer, protein inhibitors, molecular mass standards for SDS-PAGE, DMSO, TX-100, Histopaque, Sodium dodecyl sulphate (SDS), 5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide (JC-1), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and propidium iodide (PI) were obtained from Sigma-Aldrich (Munich, Germany). Tween, Sulphuric acid, acrylamide and dithiotreitol were purchased from Carl Roth GmbH, (Karlsruhe, Germany). Ammonium persulfate and N,N,N,N-tetramethylenediamine were obtained from BioRad (Munich, Germany). 3,3’,5,5’-tetramethylbenzidine was purchased from eBioscience Inc. (San Diego, USA). Following primary antibodies were used: caspase-3, poly (ADP-ribose) polymerase (PARP), claspin, survivin, bcl-2, cytochrome c (Cell Signaling Technology, Danvers, USA); p53 (Santa Cruz biotechnology, Santa Cruz, CA, USA); X-chromosome-linked IAP (XIAP) and Annexin V-APC (BD Bioscience, Heidelberg, Germany); ß-actin-peroxidase antibody (Sigma-Aldrich, Munich, Germany). The Cytotoxicity Detection Kit was purchased from Roche (Grenzach-Wyhlen, Germany).
Carbonyl cyanide 3 chlorophenylhydrazone cccp
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is a chemical compound used in laboratory settings. It functions as an ionophore, facilitating the transport of protons and other ions across cell membranes. CCCP is commonly utilized in research applications involving the study of mitochondrial function and cellular bioenergetics.
Automatically generated - may contain errors
Lab products found in correlation
Rotenone, by Merck Group (7 mentions)
Oligomycin, by Merck Group (5 mentions)
Antimycin a, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Acrylamide, by Carl Roth (2 mentions)
Antimycin, by Merck Group (2 mentions)
Facscanto flow cytometer, by BD (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Seahorse xf24 analyzer, by Agilent Technologies (2 mentions)
Phenyl arginine β naphthylamide, by Merck Group (2 mentions)
Oligomycin a, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
25 cm2 flask, by Corning (2 mentions)
68 protocols using carbonyl cyanide 3 chlorophenylhydrazone cccp
1
Apoptosis Induction Protocol
2
Cell Viability Assay for H3R Antagonists
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Human embryonic kidney (HEK)-293 cell line (ATCC CRL-1573) was kindly donated by Prof. Dr. Christa Müller (Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn). Hepatoma HepG2 (ATCC HB-8065) cell line was kindly donated by the Department of Pharmacological Screening, Jagiellonian University Medical College. The cell cultures’ growth conditions in presence of H3R antagonist 4 were applied as described before [46 (link),51 (link)]. The cells’ viability was assessed after 72 h of incubation with H3R antagonist and the following references: doxorubicin (Sigma-Aldrich) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich). CellTiter 96® AQueous non-radioactive cell proliferation assay (MTS) was purchased from Promega® and added to each well in the volume of 20 µL. The cells were then incubated for 2–5 h. The microplate reader EnSpire (PerkinElmer Ltd., Waltham, MA, USA) was used to measure the absorbance at 490 nm.
3
Mitochondrial Membrane Potential Assay
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Cells were cultured in poly-l -Lysine-coated (R&D Systems, Wiesbaden-Nordenstadt, Germany) 6-well or 96-well plates for 24 hrs and then treated with gemcitabine at 0, 25 or 50 nM for 6 or 24 hrs, respectively. Next, cells were stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyaniniodide (JC-1) solution (Sigma-Aldrich) at a final concentration of 25 μg/ml for 30 min. Fluorescence was measured employing a CytoFluor 4000 plate reader at 485 nm (excitation) and at 530 and 580 nm (emission). The ratio of green and red fluorescence signals served as surrogate readout for the mitochondrial membrane potential (Δψm) independent of the mitochondrial mass. For fluorescence imaging, cells were grown directly on slides, treated as described above and pictures taken using the Zeiss Axiovert 135 TV fluorescence microscope (Carl Zeiss). For FACS analysis, cells were harvested and analysed using the Accuri C6 Flow Cytometer® (BD Biosciences). Qualitative and quantitative data were recorded through CFlow Plus software (BD Biosciences). Valinomycin (Sigma-Aldrich) or carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich) served as negative controls. All experiments were performed at least in triplicate.
4
Mitochondrial Respiration and Ion Channel Inhibitors
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DiBAC4(3) was obtained from Thermo Fisher Scientific. Rotenone, antimycin, oligomycin B and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (all from Sigma-Aldrich, Dorset, UK) were dissolved in DMSO as 1, 1, 6 and 10 mM stock solutions, respectively. K+ channels inhibitors, glibenclamide (Sigma-Aldrich), penitrem A (Alomone labs, Jerusalem, Israel), tram34 (Sigma-Aldrich), apamin (Tocris Bioscience, Abingdon, UK) and XE991 (Tocris Bioscience) were also prepared in DMSO as 10, 1, 10, 1 and 10 mM stock solutions, respectively. Other chemicals were obtained from Sigma-Aldrich or VWR (Leicestershire, UK).
5
Assessment of Mitochondrial Membrane Potential
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The detection of changes in the inner electrochemical Δψm in living cells was performed as described previously (35 (link)), using the cationic, lipophilic JC-1 dye (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma-Aldrich; Merck KGaA), a mitochondrial potential disrupter, was used as a control. The melanoma A375 cells were pre-treated with secosteroids at a concentration of 100 nM and then exposed to 2.4 and 12 µM cisplatin or 2.0 and 10 µM dacarbazine for an additional 3 h, or to 75 nM hydrogen peroxide for 1-3 h. Following the treatment with the selected compounds, the cells were harvested and suspended in 1 ml PBS at room temperature. CCCP solution in dimethylsulfoxide (DMSO) was added to the positive control tube only (2 µM final concentration) and the cells incubated at 37°C for 5 min. JC-1 solution (2 µM in DMSO) was added to all tubes and the cells were incubated at 37°C for 15 min, then centrifuged at 1,000 × g for 10 min at room temperature, and resuspended in 500 µl PBS. The samples were kept on ice until they were analyzed on the FACSCalibur flow cytometer using the CellQuest Pro analysis software.
6
Evaluating Efflux Pump in NDM-5 K. pneumoniae
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Phenotypic evaluation of the efflux pump in NDM-5-producing K. pneumoniae was assessed by measuring the minimum inhibitory concentrations (MICs) for ciprofloxacin and meropenem before and after exposure to the efflux pump inhibitor (EPI), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, Dorset, UK) at a concentration of 20 mg/L. If the MIC values decreased 4-fold or greater in the presence of EPI, this was defined as a significant inhibition effect.22 (link)
7
Measuring Mitochondrial Membrane Potential
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Cells were seeded in 12-well plates and transfected for 24 h as described above. Afterwards, cells were incubated with 20 nM tetramethylrhodamine methyl ester (TMRM; Molecular Probes, Eugene, OR, USA) for 15 min at 37°C, harvested and analyzed by flow cytometry on a BD-FACS canto Flow Cytometer8 (link),10 (link). As positive control, mitochondrial depolarization was elicited using 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma Aldrich, St. Louis, MO, USA) for 30 min at 37°C.
8
Goat Semen Preparation for In Vitro
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All chemicals, such as tris (hydroxymethyl), citric acid, D-glucose, natrium pyruvate, 3-chloro-1,2-propanediol (3-MCPD) as a glycolysis inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as mitochondrial uncoupler were purchased from Sigma-Aldrich (St. Louis, CA, USA). Meanwhile, the ATP bioluminescence assay kit II was purchased from Roche (Mannheim, Germany).
Semen was collected from 3 fertile male Ettawa Crossbred goats using an artificial vagina. Furthermore, the semen from each buck was washed twice by centrifugation at 250 g for 5 min at RT with tris-citrate buffer (pH 7.2) to exclude seminal plasma. All animal work was performed under the approval of the Research Ethics Committee of the Universitas Padjadjaran (approval no. 39/UN6.KEP/EC/2023).
Semen was collected from 3 fertile male Ettawa Crossbred goats using an artificial vagina. Furthermore, the semen from each buck was washed twice by centrifugation at 250 g for 5 min at RT with tris-citrate buffer (pH 7.2) to exclude seminal plasma. All animal work was performed under the approval of the Research Ethics Committee of the Universitas Padjadjaran (approval no. 39/UN6.KEP/EC/2023).
9
NMDAR Signaling Pathway Analysis
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Salts, reagents, NMDA, adenosine tri-phosphate (ATP), D(-)-2-amino-5-phosphonopentanoic acid (APV), Kynurenic acid (KYNA), Dizocilpine (MK-801), XestosponginC (XesC), Ryanodine (Ry), Genistein (GX) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were from Sigma Chemical Co. (St. Louis, MO, USA). Cell culture media, supplements, master mix, Hoechst, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzim- idazolylcarbocyanine iodide (JC-1), Fluo-4 acetomethylester (Fluo-4-AM) and Lipofectamine 2000 were from Life Technologies (Carlsbad, CA, USA). Culture plates were from Costar (Tewksbury, MA, USA). Antibodies (Abs) against NMDAR subunits were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary Abs were from Jackson Immunochemicals (West Grove, PA, USA). Western Blot (WB) reagents were from Bio-Rad (Hercules, CA, USA), Enhanced Chemiluminiscent (ECL) reagents were from Thermo Scientific (Rockford, IL, USA), and Hybond and Hyperfilm were from GE Healthcare Amersham (Piscataway, NJ, USA). Tripure reagent and Protease Inhibitory cocktail were from Roche (Basel, CH). M-MLV reverse transcriptase was from Promega (Madison, WI, USA). Labeled Taq-Man probes for NMDAR subunits or 18S rRNA and siRNA were from Applied Biosystems (Foster City, CA, USA).
10
Measuring Metabolic Activity in VSMCs
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Oxygen consumption rate (OCR) was measured in 24-well plates using a Seahorse XF-24 analyzer (Seahorse Bioscience). VSMCs were serum-starved for 24 h and incubated with 10% FBS with or without melatonin (2 mM). On the day before the experiment, the sensor cartridge was placed in calibration buffer supplied by Seahorse Bioscience and incubated at 37˚C in a non-CO2 incubator for 1 h. Oligomycin (Sigma), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma), and rotenone (Sigma) were added at the indicated times during OCR measurement.
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