Cytochrome C Detection Kit

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. 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 ArrayScan® HCS Reader and Compartmental Analysis Bioapplication Software Module. Stained cells also can be imaged using fluorescence or confocal microscopy.

Figure 2. 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 3. Normal cytochrome c (top left) and redistribution of cytochrome c (top right). Dose response curve of cytochrome c in HeLa cells treated with staurosporine (bottom). Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of staurosporine).

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