H9c2 cardiomyoblast cell line was purchased from Shanghai Cell Bank (Shanghai, China). LTA was purchased from National Institutes for Food and Drug Control (Beijing, China). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s medium (DMEM) were purchased from Gibco BRL (Gaithersburg, USA). Caspase-3/9 activity assay kits and 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromid (MTT) were purchased from Sigma-Aldrich (St. Louis, USA). MDA, GSH, SOD, IL-1β, IL-12, and TNFα assay kits were purchased from Jiancheng Biological Engineering (Nanjing, China). Cytochrome-c immunoassay kit was purchased from R&D systems (Minneapolis, 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 (CA, USA). Gamma H2AX (γH2AX) antibody was purchased from BioLegend (San Diego, USA). Real-time PCR reagents were purchased from Thermo Fisher (Waltham, MA, USA). TIM was isolated and identified by Prof. Lin from Shantou University Medical College (Shantou, China) (14 ). All solvents and chemicals used in this study were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). LTA and TIM were dissolved in DMSO in the in vitro experiments.
Cytochrome c immunoassay kit
Manufactured by R&D Systems
Sourced in United States
The Cytochrome-c immunoassay kit is a laboratory tool designed to detect and quantify the presence of cytochrome-c, an important cellular protein, in biological samples. This kit utilizes an antibody-based approach to measure cytochrome-c levels.
Automatically generated - may contain errors
Lab products found in correlation
5 5 6 6 tetrachloro 1 1 3 3 tetraethylbenzimidazolylcarbocyanine iodide jc 1, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Real time pcr reagents, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescin diacetate dcfh da, by Thermo Fisher Scientific (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Enoxacin, by Merck Group (1 mentions)
Taxol, by Merck Group (1 mentions)
Wpmy 1, by Shanghai Cell Bank (1 mentions)
3 3 dihexyloxacarbocyanine iodide dioc6, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Hepg2, by Shanghai Cell Bank (1 mentions)
Mcf 7, by Shanghai Cell Bank (1 mentions)
Lncap, by Shanghai Cell Bank (1 mentions)
Du145, by Shanghai Cell Bank (1 mentions)
Panc 1, by Shanghai Cell Bank (1 mentions)
Sk mes 1, by Shanghai Cell Bank (1 mentions)
4 protocols using cytochrome c immunoassay kit
1
Cardiomyoblast Stress Response Protocol
2
Cytoplasmic Cytochrome c Quantification
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LECs were plated in cell culture dishes (5×106 cells/60 cm2) and treated the next day with TSA as aforementioned. After 24 h, cytoplasmic extracts were obtained by digitonin permeabilization. Briefly, cells were trypsinized (0.125% trypsin/0.1% EDTA) for 5 min and centrifuged at 259 g for 10 min, and the pellet was resuspended in 250 μl PBS. Permeabilization of the membranes was obtained by adding 250 μl digitonin/sucrose (80 mg/ml, Fluka, Buchs, Switzerland) for 30 s. Then, samples were centrifuged for 1 min at 14000 g [12 (link)], and supernatants with equal amounts of protein (protein determination by DC protein assay; BioRad, Munich, Germany) were used in a commercial cytochrome c immunoassay kit (R&D Systems, Wiesbaden, Germany). The assay was performed according to the manufacturer’s manual. Optical density (450 nm) was measured using an ELISA reader (ELISA-Reader ASYS Expert 96, Deelux Labortechnik, Gödenstorf, Germany).
3
Cytochrome c Release Assay in A549 Cells
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A549 cells were seeded in 2 ml fresh medium at an initial density of 1 × 106 cells/ml and incubated up to 4 hours with or without 20 μM MPD. After the incubation, the cells were harvested by centrifugation and washed twice with PBS. The cells were suspended in 200 μl lysis buffer (195 mM mannitol; 65 mM sucrose; 2 mM HEPES, pH 7.4; 0.05 mM EGTA; 0.01 mM MgCl2; 0.5 mg/ml BSA) and lysed by the addition of 0.01% digitonin. The cytosolic fraction was obtained from 10,000 × g centrifugation for 10 minutes and was collected for cytochrome c assay in 1 × RD5P calibrator diluent (cytochrome c Immunoassay Kit; R and D Systems, MN, USA).
4
Apoptosis Induction in Cancer Cell Lines
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Normal human prostate/stroma cell line WPMY-1, lung adenocarcinoma cell line A549, pancreatic adenocarcinoma cell line PANC-1, hepatocellular carcinoma cell line HepG2, prostate adenocarcinoma cell line PC-3, prostate carcinoma cell lines DU145, LNCaP and C4-2, glioblastoma cell line U251, squamous cell carcinoma cell line SK-MES-1, breast adenocarcinoma cell line MCF-7, and osteosarcoma cell line MG-63 were purchased from Shanghai Cell Bank (Shanghai, China). Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, USA). 3,3′-dihexyloxacarbocyanine iodide (DiOC6) was purchased from Sigma (St. Louis, MO, USA). Caspase-3/9 colorimetric assay kits and cytochrome-c immunoassay kit were purchased from R&D Systems (Minneapolis, MN, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was purchased from Molecular Probes (CA, USA). Annexin V/PI apoptosis assay kit was purchased from GeneCopoeia (Rockville, MD, USA). PCR reagents were purchased from Thermo Fisher (Waltham, MA, USA). Taxol and Enoxacin were purchased from Sigma (St. Louis, MO, USA). All solvents were purchased from Sinopharm Co. Ltd. (Shanghai, China). Enoxacin was dissolved in PBS for in vitro and in vivo experiments.
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