Cellomics HCS Reagent Kit for detection cytochrome c. | | | | | normal
cytochrome c | treat cells | redistribution of
cytochrome c | analyze | conclude |
DescriptionThe Cellomics Cytochrome C Detection Kit measures release of cytochrome c from mitochondria, a key early step in apoptosis or programmed cell death. The kit contains a mouse monoclonal anti-cytochrome c primary antibody, a secondary antibody conjugated to DyLight 549 Fluorophore, and various other reagents and buffers required for immunofluorescent detection of cytochrome c for high-content screening (HCS) assays. Cytochrome c is a small (12 kDa) heme protein present on the inner mitochondrial membrane and is critical for oxidative phosphorylation and production of ATP in cells and the mitochondrial pathway of apoptosis. Upon apoptosis stimulation, cytochrome c is released from mitochondria and then associates with caspase 9/Apaf-1 to form a complex. This complex formation leads to caspase 9 and caspase 3 activation and nuclear fragmentation. To measure cytochrome c levels, HeLa cells were treated with staurosporine (Figure 1). In normal cells cytochrome c is in the mitochodria, which can be detected as cytoplasmic spots. Upon apoptosis induction, cytochrome c is released from the mitochondria and diffuses into the nucleus. The assay was developed and optimized using the Thermo Scientific ArrayScan HCS Reader and Compartmental Analysis Bioapplication Software Module. Stained cells also can be imaged using fluorescence or confocal microscopy. | Figure 1. Staining of cytochrome c in HeLa cells treated with or without staurosporine (1 µM) for 24 hours. Cells were stained according to the kit protocol and imaged using the ArrayScan HCS Reader. Staurosporine treatment leads to cytochrome c release from mitochondria and increased nuclear staining in treated cells. |
| Figure 2. Dose response curve of cytochrome c in HeLa cells treated with staurosporine. Data represents mean ±SD from three plates (eight wells per 96-well plate per dose of staurosporine). |
Related Poster Presentations | Bhaskar S. Mandavilli, Krishna Vattem, and Richik N. Ghosh (2008) Automated, Quantitative, Multi-Parametric Monitoring of Different Programmed Cell Death Pathways Using Cell-Based High-Content Screening Assays. | | Kay Opperman, Bhaskar S. Mandavilli, and Richik N. Ghosh (2008) Assessment of Oxidative Stress and DNA Damage in Individual Cells Using Automated, Quantitative Cell-Based Imaging. |
Kit Contents| Component | 8405601 | 8405602 | | Cytochrome C Primary Antibody | 15 µl | 75 µl | | DyLight 549 Conjugated Goat Anti-Mouse IgG | 30 µl | 72 µl | | Hoechst Dye | 30 µl | 30 µl | | Wash Buffer (10X Dulbecco's PBS) | 100 ml | 100 ml | | Wash Buffer II (10X D-PBS with Tween®-20) | 100 ml | 100 ml | Permeabilization Buffer (10X Dulbecco's PBS
with 1% Triton® X-100) | 100 ml | 100 ml | | Blocking Buffer (10X) | 85 ml | 85 ml | | Thin Plate Seal Assembly | 7/pack | 7/pack |
Ordering InformationScreening size kits and components (bulk and custom) available with special pricing. Please call. To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products. Choose "Order Online" in table to see pricing and order online (U.S. only). | Buy | Product # | Description | Pkg. Size | | Order Online | 8405601 | Cytochrome C Detection Kit | 1 x 96 | | Order Online | 8405602 | Cytochrome C Detection Kit | 5 x 96 |
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