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CBA077 InnoCyte™ Flow Cytometric Cytochrome c Release Kit

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CBA077
  
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Overview

Key Spec Table

Detection Methods
Fluorescence
Description
Overview

This product has been discontinued.





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.
Catalogue NumberCBA077
Brand Family Calbiochem®
Application Data
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).

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-α Mouse mAb (7H10BD4F9) (Cat. AP1036). The F1F0-α 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-α 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-α, right panel) were generated using FCS Express (DeNovo Software).

The results from Figure 2 were analyzed with FCS Express (DeNovo Software). The percentage of cells positive for cytochrome c and F1F0-α are shown.
Materials Required but Not Delivered• Phosphate Buffered Saline (PBS; e.g. PBS Tablets, Cat. No. 524650)
• Paraformaldehyde (8% in PBS)
• Vacuum and vacuum flask for aspiration of wash buffer
• Fluorescent microscope equipped for FITC detection or flow cytometer equipped with appropriate laser for detection of FITC (i.e., 488 nm argon laser)
• Apoptosis inducer (i.e., Actinomycin D, Cat. 114666, or Camptothecin, Cat. 208925)
• DAPI, 100 µg/ml stock (Cat. No. 268298)
References
ReferencesGreen, 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.
Product Information
Detection methodFluorescence
Form50 Cell stainings
FormatFlow Cytometry, Immunofluorescence Microscopy
Kit containsPermeabilization Buffer, Blocking Buffer, 10X Wash Buffer, Anti-Cytochrome c, Anti-IgG FITC, and a user protocol.
Applications
Application ReferencesKing, M.A., et al. 2007. Cytometry, Part A 71A, 668.
Key Applications Flow Cytometry
Immunofluorescence
Biological Information
Sample TypeIntact Cells
Product Usage Statements
Intended useThe Calbiochem® InnoCyte™ Flow Cytometric Cytochrome C Release Kit is suitable for assessing the relocalization of cytochrome c from the mitochondria to the cytoplasm. The kit can be used for inhibitor screening and for assessing the regulation of apoptotic signaling in cells.
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Standard Handling
Storage -20°C
Storage ConditionsUpon arrival store the entire contents of the kit at -20°C. Avoid freeze/thaw cycles of the antibodies.
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Supplemental Information
Kit containsPermeabilization Buffer, Blocking Buffer, 10X Wash Buffer, Anti-Cytochrome c, Anti-IgG FITC, and a user protocol.
Global Trade Item Number
Catalogue Number GTIN
CBA077 0