The cardiomyoblast cell line H9c2 was purchased from Beijing Cell bank (Beijing, China). Fetal bovine serum and RPMI-1640 medium were from Gibco BRL (Gaithersburg, MD, USA). LTA and 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Caspase-3/9 activity assay kits were from Promega (Madison, WI, USA). Glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), IL-1β, and TNF-α assay kits were purchased from Jiancheng Biological Engineering (Nanjing, China). The cytochrome c assay kit was from R&D systems (Minneapolis, MN, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR, USA). Real-time PCR reagents were purchased from Thermo Fisher (Waltham, MA, USA). DHAG was provided by Prof. Teng from Zhengzhou University.15 All solvents and chemicals were analytical grade and were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). LTA and DHAG were dissolved in dimethyl sulfoxide (DMSO).
Cytochrome c assay kit
Manufactured by R&D Systems
Sourced in United States
The Cytochrome c Assay Kit is a quantitative colorimetric assay designed to measure cytochrome c levels in cell and tissue lysates. The kit uses a proprietary detection reagent that binds to cytochrome c, producing a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
5 5 6 6 tetrachloro 1 1 3 3 tetraethylbenzimidazolylcarbocyanine iodide jc 1, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescin diacetate dcfh da, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Real time pcr reagents, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Cytosol extraction buffer, by Abcam (1 mentions)
Caspase 3 assay kit, by Merck Group (1 mentions)
3 protocols using cytochrome c assay kit
1
Cardioprotective Effects of DHAG on H9c2 Cells
2
Mitochondrial Cytochrome c Release Assay
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To determine mitochondrial cytochrome c release, the cytosolic fractions of the cells were first separated from the mitochondrial fractions according to the manufacturer’s procedures (BioVision, USA). Next, the cells were exposed to DOX-TRF or DOX for 6, 12 or 24 h and collected by centrifugation at 600 g for 5 min at 4 °C. After homogenizing the cell pellets with cytosol extraction buffer (BioVision, USA) on ice for 40 min, the supernatant (cytosolic protein fraction) was immediately stored at −70 °C until use for biochemical analysis.
The level of cytochrome c in the cytosolic fraction was measured using a Cytochrome c Assay kit (R&D Systems, USA). This colorimetric assay was performed in clear 96-well plates pre-coated with an anti-cytochrome c monoclonal antibody, according to the manufacturer’s instructions [20 (link)]. Finally, the plates were read at 450 nm using a Cary 50 microplate reader (Cary, Melbourne, Australia).
The level of cytochrome c in the cytosolic fraction was measured using a Cytochrome c Assay kit (R&D Systems, USA). This colorimetric assay was performed in clear 96-well plates pre-coated with an anti-cytochrome c monoclonal antibody, according to the manufacturer’s instructions [20 (link)]. Finally, the plates were read at 450 nm using a Cary 50 microplate reader (Cary, Melbourne, Australia).
3
Rhodamine 123 and Caspase-3 Assay Protocol
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Rhodamine 123 (Rh 123), 2,7‐dicloroflurescein diacetate (DCHF‐DA), and caspase-3 assay kit were purchased from Sigma Chemical Co (St. Louis, MO). Cytochrome c assay kit was purchased from R&D Systems, Inc. (Minneapolis, MN). Also, other chemicals were of the highest commercial grade available.
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