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| | InnoCyte™ Flow Cytometric Cytochrome c Release Kit |
|  | Cat. No. CBA077 |
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| Format: Flow Cytometry, Immunofluorescence Microscopy | | Form: 50 Cell stainings | | Detection Method: Fluorescence | | Sample Type: Intact Cells | | Kit Contents: Permeabilization Buffer, Blocking Buffer, 10X Wash Buffer, Anti-Cytochrome c, Anti-IgG FITC, and a user protocol. | | Comments: A convenient and sensitive assay for determination of the relocalization of cytochrome c from the mitochondria to the cytoplasm using flow cytometry or fluorescent microscopy. The kit provides a rapid method for inhibitor screening and assessing the regulation of apoptotic signaling in cells. It relies on the selective permeabilization of the cellular membrane for release of cytosolic components while leaving the mitochondrial membrane intact. | | Ref.: Green, D.R. and Kroemer, G. 2004. Science 305, 626. Jurgensmeier, J.M., et al. 1998. Proc. Natl. Acad. Sci. USA 95, 4997. Luo, X., et al. 1998. Cell 94, 481. Kluck, R.M., et al. 1997. Science 275, 1132. Li, P., et al. 1997. Cell 91, 479. Liu, X., et al. 1996. Cell 86, 147. |
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Analysis of Cytochrome c Staining by Flow Cytometry. Jurkat cells (4 x 105/ml) were either left untreated (black) or treated (red) with 1 µM actinomycin D (Cat. No. 114666) for 12 h (please visit www.calbiochem.com to see the color version of this figure. Cells (1 x 106) were processed as outlined in Protocol A, including the following controls: cells were double-stained with the Anti-Cytochrome c antibody and Anti-F1F0-a Mouse mAb (7H10BD4F9) (Cat. AP1036). The F1F0-a ATPase of Complex V is not lost from apoptotic mitochondria and is localized to the mitochondrial inner membrane. Isotype specific secondary antibodies were used to discriminate between cytochrome c positive cells (Anti-IgG1 FITC) and F1F0-a ATPase positive cells (Anti-IgG2b PE). Isotype controls were performed for each sample to ensure specific staining (data not shown). Ten thousand events were acquired with a FACScan flow cytometer (Becton Dickinson). Histograms for FL1, FITC (cytochrome c, left panel) and FL2, PE (F1F0-a, right panel) were generated using FCS Express (DeNovo Software).
Cytochrome c Staining of Treated and Untreated Cells. Jurkat cells (4 x 105/ml) were either left untreated (left panel) or treated (right panel) with 1 µM actinomycin D (Cat. No. 114666) for 8 h. Cells (1 x 106) were processed as outlined in Protocol A (through step 12) and subsequently prepared for fluorescence microscopy as outlined in Protocol B. Nuclei stained with DAPI (blue) and cytochrome c stained with FITC (green) (please visit www.calbiochem.com to see the color version of this figure) were visualized using a Nikon Eclipse E600 microscope and a 40X dry objective. Cytochrome c is retained in the mitochondria in viable cells (arrows, left panel) and is lost from cells treated with actinomycin D (right panel).
Results from Figure 2 Analyzed by FCS Express. The results from Figure 2 were analyzed with FCS Express (DeNovo Software). The percentage of cells positive for cytochrome c and F1F0-a are shown.
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