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CBA077 InnoCyte™ Flow Cytometric Cytochrome c Release Kit

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      Overview

      Replacement Information

      Key Specifications 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
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      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
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsPermeabilization Buffer, Blocking Buffer, 10X Wash Buffer, Anti-Cytochrome c, Anti-IgG FITC, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalog Number GTIN
      CBA077 0

      Documentation

      InnoCyte™ Flow Cytometric Cytochrome c Release Kit Certificates of Analysis

      TitleLot Number
      CBA077

      References

      Reference overview
      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.
      User Protocol

      Revision19-September-2008 RFH
      Form50 Cell stainings
      FormatFlow Cytometry, Immunofluorescence Microscopy
      Detection methodFluorescence
      StorageUpon arrival store the entire contents of the kit at -20°C. Avoid freeze/thaw cycles of the antibodies.
      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.
      BackgroundThe basic molecular framework for regulating and executing cell death is conserved across phylogeny. Apoptotic cell death is characterized by diverse initiating signals and multiple death-regulating members of both the caspase and bcl-2 gene families. In what has been termed the intrinsic cell death pathway, caspases function within a proteolytic cascade that is punctuated by members of the Bcl-2 family. Bcl-2 family members regulate caspase-9 activation through the compartmentalization of cytochrome c. Although there are different inducers of cell death, there exists a common commitment step to the cell death process (point of no return) that coincides with the release of cytochrome c from mitochondria. Cytochrome c is encoded by a nuclear gene and translated as apocytochrome c. Only upon transport to the mitochondrial intermembrane space does cytochrome c, loaded with heme, fulfill its function as an electron carrier in oxidative phosphorylation, shuttling electrons from Complex III (cytochrome c reductase) to Complex IV (cytochrome c oxidase). Upon induction of apoptosis, holocytochrome c is released from mitochondria and associates with Apaf1 and procaspase-9, forming the "apoptosome". Apoptosome formation results in activation of caspase-9. Caspase-9 is then responsible for activation of the proteolytic caspase cascade and the ultimate demise of the cell. The analysis of cytochrome c release is important in determining the commitment to apoptosis in model signaling systems. Widely employed cellular fractionation strategies for determination of cytochrome c release generally involve mechanical disruption of cells resulting in incomplete or excessive rupturing of cells and mitochondria. Subsequent analysis of subcellular fractionations by Western blot can only be regarded as qualitative, reflecting the quality of the fractionation procedure. In contrast the InnoCyte™ Cytochrome C Release Kit is used for determination of mitochondrial cytochrome c localization on a per cell basis.
      Principles of the assayThe Calbiochem® InnoCyte™ Flow Cytometric Cytochrome C Release Kit relies on the selective permeabilization of the cellular membrane for release of cytosolic components while leaving the mitochondrial membrane intact. Viable cells will display mitochondrial staining of cytochrome c while cells committed to the apoptotic process do not stain since they release cytochrome c from the mitochondria to the cytosol. Cytochrome c is detected with a monoclonal antibody specific to cytochrome c and a FITC-labeled secondary antibody either by flow cytometry or fluorescent microscopy. Other fluorescent-labeled antibodies can be substituted for the FITC-labeled secondary making the kit amenable to multiparameter analysis of apoptotic cells (see figure 2). The cytosolic cytochrome c signal in apoptotic cells is eliminated using the selective permeabilization step.
      Materials provided• 10X Wash Buffer (Kit Component No. JA9364-50ML): 1 bottle, 50 ml, contains BSA
      • Permeabilization Buffer, (Kit Component No. JA9362-15ML): 1 vial, 15 ml
      • Anti-Cytochrome C (Kit Component No. JA9365-15UG): 1 vial, 15 µg, supplied at 500 µg/ml, contains 0.09% sodium azide
      • Anti-IgG FITC (Kit Component No. JA9366-100UL): 1 vials, 100 µl, contains BSA
      • Blocking Buffer (Kit Component No. JA9363-50ML): 1 bottle, 50 ml, contains BSA
      Materials Required but not provided• 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)
      Precautions and recommendations• A time course must be performed to determine when cytochrome c is lost from the mitochondria. Results will vary depending on cell type and death stimulus.
      • The kit has been optimized for use with the included anti-IgG FITC, however, other secondary antibodies such as IgG isotype specific FITC and PE-labeled antibodies have been used successfully (see figure 2).
      Reagent preparationPrepare all reagents immediately prior to use. The following table provides reagent preparation instructions to obtain the volume of Working Solution (WS) required for 5 samples:

      Table 1: Reagent Preparation
      Detailed protocolA. Protocol for detection by flow cytometry:

      1. Grow cells to an appropriate density and treat with apoptosis inducer. For example, grow 2 x 20 ml Jurkat cells in T75 flasks to a density of 4 x 105 cells/ml. Treat one of the flasks with 1 µM Actinomycin D for 8 h.
      Note: appropriate cell density and treatment time will vary among cell types and apoptosis inducers, so conditions should be optimized for each cell type and inducer combination.
      2. Harvest the cells in 50 ml conical tubes by centrifugation at 1000 g for 5 min in a clinical centrifuge.
      3. Remove the culture supernatant from each tube by aspirating the liquid with a pasteur pipette connected to a vacuum source. Using a clean, sterile pipet, gently resuspend the cell pellet(s) in 10 ml PBS. Remove 20 µl from the cell suspension and count the number of cells using a hemocytometer. Using a clean, sterile pipet, transfer 1-3 x 106 cells to a 15 ml conical tube and centrifuge as outlined in step 2.
      4. Remove the PBS from each tube by aspirating the liquid with a pasteur pipette connected to a vacuum source. Using a clean, sterile micropipette tip, gently resuspend the cell pellet(s) in 300 µl Permeabilization Buffer. Incubate for 10 min on ice.
      5. Fix the cells by adding 300 µl 8% paraformaldehyde in PBS directly to the tubes. Gently mix the cells by inverting tubes 5 times. Incubate for 20 min at room temperature.
      6. Pellet the cells by centrifugation using the conditions outlined in step 2.
      7. Wash the cells by adding 1 ml 1X Wash Buffer to each tube, followed by centrifugation using the conditions outlined in step 2. Remove the 1X Wash Buffer from each tube by aspirating the liquid with a pasteur pipette connected to a vacuum source. Using a clean, sterile micropipette tip add 1 ml 1X Wash Buffer to each tube and repeat the process for a total of 3 washes. After the final wash, take care not to disturb cell pellet (cells may be difficult to see) when aspirating the 1X Wash Buffer.
      8. Add 250 µl Blocking Buffer to each tube and gently resuspend the cells by pipetting up and down. Incubate for 1 h at room temperature.
      9. Add 250 µl Anti-Cytochrome C WS to each tube and gently mix cells by inverting the tube 5 times. Incubate for 1 h at room temperature.
      10. Wash the cells 1 time with 1X Wash Buffer as outlined in step 7.
      11. Add 500 µl Anti-IgG FITC WS to the cells. Incubate for 1 h at room temperature in the dark.
      12. Wash the cells 1 time with 1X Wash Buffer as outlined in step 7.
      13. For detection by fluorescence microscopy proceed to Protocol B.
      14. For detection by flow cytometry, gently resuspend the cells in 500 µl 1X Wash Buffer.
      15. Analyze cells using a flow cytometer capable of assessing FITC staining (i.e., 488 nm argon laser).

      B. Protocol for detection by fluorescent microscopy:

      1. For detection by fluorescence microscopy, gently resuspend the cell pellet from step 12 above in 100 µl 1X Wash Buffer.
      2. Add 1 µl DAPI (100 µg/ml stock) to the cells.
      3. Incubate for 10 min on ice in the dark.
      4. Place a single drop of cell suspension on a standard microscope slide and cover by very gently dropping a round cover slip onto the sample.
      5. View under a fluorescent microscope using the 10X or 40X dry objective.
      Example data

      Figure 1: 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).

      Figure 2: 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-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).

      Figure 3: 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-α are shown.
      Application referencesKing, M.A., et al. 2007. Cytometry, Part A 71A, 668.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      InnoCyte™ is a trademark of EMD Chemicals, Inc.