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| Kit Size | 25, 100 |
|---|---|
| Caspase | Poly Caspase, Caspase 3/7, Caspase 8, Caspase 9, Caspase 1 |
Caspase (poly, 3/7, 8, 9, 1) & Mitochondria Membrane Potential Detection – MitoCasp
Caspase enzymes specifically recognize a 4 amino acid sequence (on their substrate) which necessarily includes an aspartic acid residue. This residue is the target for the cleavage reaction, which occurs at the carbonyl end of the aspartic acid residue(6). Caspases can be detected via immunoprecipitation, immuno-blotting techniques using caspase specific antibodies, or by employing fluorogenic substrates which become fluorescent upon cleavage by the caspase. MitoCasp uses a novel approach to detect active caspases (7-9). The methodology is based on carboxyfluorescein (FAM) labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases. These inhibitors are cell permeable and non-cytotoxic. Once inside the cell, the inhibitor binds covalently to the active caspase (10). Cells that contain bound inhibitor can be analyzed by flow cytometry or fluorescence microscopy.
Cell Technology utilizes a cationic dye to visualize mitochondrial membrane potential (15-17). The cationic dye is cell permeable and has a strong fluorescent signal in the red region and exhibits low membrane potential independent (non specific) binding and toxicity. In healthy cells the cationic dye is accumulated by the mitochondria in proportion to the DeltaPsi (membrane potential). In most cell lines, accumulation of the cationic dye in the mitochondria results in a higher fluorescence intensity. In apoptotic cells, where the mitochondrial membrane potential is compromised, the cationic dye does not accumulated in the mitochondria and these cells exhibit a lower fluorescence signal. Utilizing these two reagents in combination Caspase activity and mitochondrial membrane potential can be analyzed simultaneously. Citations Identification of single-domain, Bax-spec
Jurkat cells were stimulated with Staurosporine for 3 hours (B) or DMSO (A). The cells were then stained with the MitoCasp kit according to the protocol. The cells were then washed twice and analyzed by flow cytometry: Ex:488nm Em: FL1 and FL2.
Fig A. Healthy cells show a strong red fluorescence indicating intact mitochondria and no green fluorescence, indicating no active caspases.
Fig B. Apoptotic cells show a loss of red fluorescence (y axis) indicating loss of mitochondrial membrane potential and positive green fluorescence (x axis) indicating active caspases.
| Document Title |
| MitoCaspProtocol |
| MitoCasp Datasheet |
| msds.MitoCasp |
| Title | File | Link | Author(s) | Journal | Year; Edition:Pages |
| Selective cytotoxicity of Pancratistatin-related natural Amaryllidaceae alkaloids: evaluation of the activity of two new compounds | http://www.cancerci.com/content/7/1/10 | Griffin, Sharda, Sood, Nair, et al | Cancer Cell Int | V7 2007 | |
| OSU-03012, a Novel Celecoxib Derivative, Is Cytotoxic to Myeloma Cells and Acts through Multiple Mechanisms | http://clincancerres.aacrjournals.org/cgi/content/abstract/13/16/4750 | Zhang, Suvannasankha, Crean, et.al | Clinical Cancer Research | 13, pp 4750-4758, Aug 15, 2007 | |
| Galectin-3 but not galectin-1 induces mast cell death by oxidative stress and mitochondrial permeability transition | http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T20-4RTM2SR-2&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ed18c5ccefa05603a620115964776eea | Yoshihiro Suzuki, Toshio Inoue, Tetsuro Yoshimaru and Chisei Ra | Biochimica et Biophysica Acta (BBA) - Molecular Cell Research | Vol 1783, issue 5, pp 924-934, 2008 | |
| Nitric oxide protects mast cells from activation-induced cell death: the role of the phosphatidylinositol-3 kinase-Akt-endothelial nitric oxide synthase pathway | http://www.jleukbio.org/content/83/5/1218.short | Toshio Inoue, Yoshihiro Suzuki, Tetsuro Yoshimaru and Chisei Ra | Journal of Leukocyte Biology | vol. 83 no. 5, pp1218-1229, 2008 doi:10.1189/jlb.1007667 |
| Reference |
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| Walker, N. P., R. V. Talanian, K. D. Brady, L. C. Dang, N. J. Bump, C. R. Ferenz, S. Franklin, T. Ghayur, M. C. Hackett and L. D. Hammill. 1994. Crystal Structure of the Cysteine Protease Interleukin-1ß-Converting Enzyme: A (p20/p10)2 Homodimer. Cell 78:343-352. |
| Wilson, K. P., J. F. Black, J. A. Thomson, E. E. Kim, J. P. Griffith, M. A. Navia, M. A. Murcko, S. P. Chambers, R. A. Aldape, S. A. Raybuck, and D. J. Livingston. 1994. Structure and mechanism of interleukin-1 beta converting enzyme. Nature 370: 270-275. |
| Rotonda, J., D. W. Nicholson, K. M. Fazil, M. Gallant, Y. Gareau, M. Labelle, E. P. Peterson, D. M. Rasper, R. Ruel, J. P. Vaillancourt, N. A. Thornberry and J. W. Becker. 1996. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nature Struct. Biol. 3(7): 619-625. |
| Kumar, S. 1999. Mechanisms mediating caspase activation in cell death. Cell Death and Differ. 6: 1060-1066. |
| Thornberry, N. A., T. A. Rano, E. P. Peterson, D. M. Rasper, T. Timkey, M. Garcia-Calvo, V. M. Houtszager, P. A. Nordstrom, S. Roy, J. P. Vaillancourt, K. T. Chapman and D. W. Nicholson. 1997. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272(29): 17907-17911. |
| Amstad, P.A., G.L. Johnson, B.W. Lee and S. Dhawan. 2000. An in situ marker for the detection of activated caspases. Biotechnology Laboratory 18: 52-56. |
| Bedner, E., P. Smolewski, P.A. Amstad and Z. Darzynkiewicz. 2000. Activation of caspases measured in situ by binding or fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Research 259: 308-313. |
| Smolewski, P., E. Bedner, L. Du, T.-C. Hsieh, J. Wu, J. D. Phelps and Z. Darzynkiewicz. 2001. Detection of caspase activation by fluorochrome-labeled inhibitors: multiparameter analysis by laser scanning cytometry. Cytometry 44: 73-82. |
| Ekert, P. G., J. Silke and D. L. Vaux. 1999. Caspase inhibitors. Cell Death and Differ. 6:1081-1086. |
| Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., and Martinou, J.C. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144 (5): 891-901 (1999). |
| Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R. J., Matsuda, H., and Tsujimoto, Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc. Natl. Acad. Sci. USA 95: 14681-14686 (1998). |
| Basanez, G., Nechushtan, A., Drozhinin, O., Chanturiya, A., Choe, E., Tutt, S., Wood, K. A., Hsu, Y. T., Zimmerberg, J., and Youle, R. J. Bax , but not Bcl-XL decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. USA 96: 5492-5497 (1999). |
| Luo, X., Budihardio, I., Zou, H., Slaughter, C., and Wang, X. Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490 (1998). |
| Ehrenberg B, Montana V, Wei MD, Wuskell JP, Loew LM. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J. 1988 May;53(5):785-94. |
| Farkas DL, Wei MD, Febbroriello P, Carson JH, Loew LM. Simultaneous imaging of cell and mitochondrial membrane potentials. : Biophys J. 1989 Dec;56(6):1053-69. Erratum in: Biophys J 1990 Mar;57(3):following 684. |
| Russell C. Scaduto, Jr. and Lee W. Grotyohann. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J. 1999 Jan;76(1 Pt 1):469-77. |
| Rajagopal A, Pant AC, Simon SM, Chen Y. In vivo analysis of human multidrug resistance protein 1 (MRP1) activity using transient expression of fluorescently tagged MRP1. Cancer Res. 2002 Jan 15;62(2):391-6. |
| Part# | Reagent | Temperature |
| Part # 4015 | Mitochondrial Memberane Potential Cationic Dye | 2-8C |
| Refer to Product Datasheet | Caspase Detection Reagent (Poly Caspase, Caspase 3/7, Caspase 1, Caspase 8 or Caspase 9) | 2-8C |
| Part # 3028 | 10X Wash Buffer | 2-8C |
| Part # 3032 | 1X Dilution Buffer | 2-8C |
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