Cytochrome C Assay

Cytochrome C Assay

Millipore’s HCS236 Cytochrome C Assay provides a complete solution for identifying and quantifying the mitochondrial presence and cytoplasmic release of cytochrome c via cellular imaging. The reagents in the kit have been specifically optimized for HCS applications. The assay is designed to enable visualization and quantitative detection of cytochrome c, facilitating the identification and characterization of inducers and inhibitors of cytochrome c release. Key applications of this assay include apoptotic pathway analysis, neuroprotection studies, chemotherapeutic efficacy screening and in vitro toxicology applications. The nuclear dye (Hoechst 33342) may be used for measurements of cell number, DNA content and nuclear size. Additionally, the assay can be multiplexed with other probes for analysis of proximal cellular events, e.g., for upstream or downstream signaling pathway profiling or for simultaneous screening for cytotoxicity.

Cytochrome c plays a key role in regulating apoptosis. When a cell detects an apoptotic stimulus, such as DNA damage or metabolic stress, the intrinsic apoptotic pathway is triggered, and mitochondrial cytochrome c is released into the cytosol, triggering a series of events leading to apoptotic cell death. Mitochondria play a central role in cancer cell biology because tumor cells rely on glycolysis, but also because evasion of apoptosis is one of the hallmarks of cancer. Given the importance of mitochondrial events during the initial critical stages of the execution of the apoptotic program, targeting mitochondria to induce apoptosis of malignant cells is an important novel therapeutic strategy. In recent years, extensive research has focused on screening for chemical compounds, small molecules and peptides that could target the mitochondria. As a key regulator of apoptosis, cytochrome c release is widely used as a measurement in drug discovery approaches targeting apoptosis. In the context of anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells, and suppression of mitochondrial stages of apoptosis in surrounding normal cells may represent a promising paradigm for new effective therapies. The use of microscopy has led to many advances in the field of apoptosis, especially with respect to studying the mitochondrial pathway. The first descriptions of apoptosis described the morphological changes that consistently occur during this form of cell death – namely cell shrinkage, nuclear condensation, blebbing and the formation of apoptotic bodies, each of which are amenable to High Content Screening. Through the use of fluorescent antibody-based detection of cytochrome c, the role and regulation of this important protein in the events that occur during mitochondrial apoptosis may be observed and quantified.

• High Content Analysis
• Immunofluorescence
• Immunocytochemistry

Apoptosis, drug discovery, High Content Analysis, Immunocytochemistry

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

• High-Content Screening
• High-Content Analysis

Sufficient reagents for 5 X 96 well plates

• 1. Mouse anti-Cytochrome C HCS Primary Antibody, 100X
• 2. HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X
• 3. Hoechst HCS Nuclear Stain, 200X
• 4. HCS Fixation Solution with Phenol Red, 2X
• 5. HCS Immunofluorescence Buffer, 1X
• 6. HCS Wash Buffer, 1X
• 7. Staurosporine, 2.5 mM in DMSO, 250X
• 8. Paclitaxel, 0.25mM in DMSO, 250X
• 9. DMSO for Compound Serial Dilution
• 10. Compound Dilution Buffer
• 11. Plate Sealers

Store kit components under the conditions indicated on the labels. Discard any remaining reagents after 6 months.

Fluorescent

1. Sterile, tissue culture-treated black/clear bottom microplates suitable for High-Content Imaging.

2. Cell-type for assay, e.g., HeLa (human cervical adenocarcinoma, ATCC #CCL-2), A549 (human lung carcinoma, ATCC #CCL-185) or HepG2 (human hepatocellular carcinoma, ATCC #HB-8065).

3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.

4. HCS imaging/analysis system, e.g., GE Healthcare IN Cell Analyzer 1000 with Investigator software. System must be equipped with beam-splitters and filters capable of reading emission spectra in the blue and red ranges.