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FAM-FMK Pouch Cell Technology Caspase / Apoptosis Detection
FAM-FMK
Carboxyfluorescein Caspase Detection Kits™: Apoptosis detection and in situ labeling of active caspase in live cells.

$175.00$525.00

in stock
Product code: FAM
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Readable Documents

Product Description

Key Benefits

  • Non-cytotoxic assay arrests further apoptotic activity via caspase inhibition.
  • Cell permeablity permits direct visualization of cytosolic apoptotic events.
  • Apoptotic cell population does not diminish over time.
  • Add reagent directly to cells. No special buffer or media needed. No preparation of cell lysates required. Simple wash procedure.
  • Works in diverse cell lines: human, rodent, Drosophila.
  • Can be performed in conjunction with Annexin staining, TUNEL, antibody staining, or with other APO LOGIX reagents on the same population of cells.
  • Permits high through-put screening. Protocol can be adapted for ex vivo as well as in situ experiments.
  • Yields both quantitative and qualitative results. Gives strong signal with little background noise.
  • Mark activity across the range of caspase proteins. Poly-caspase and caspase-specific assays to target caspases 1, 2, 3, 6, 8, 9, or 10 are available.
  • Applications – Flow Cytometry, Fluorescence Plate Reader, Fluorescent Microscopy

Assay Principle

apop-1a
apop-1bAPO LOGIX Carboxyfluoroscein Caspase Detection Kits label active caspases in living cells undergoing apoptosis. Cell Technology’s probes utilize carboxyfluorescein(FAM)-labeled peptide fluoromethyl ketone (FMK) caspase inhibitors (FAM-peptide-FMK). These FAM-peptide-FMK compounds are both cell permeable and non-cytotoxic during the course of the assay and thus allow the detection of active caspases in living cell systems.

Fig A. Jurkat cells treated with DMSO. Cells were labeled with FAM-VAD-FMK for 1 hour. Caspase activity was detected using flow cytometry.

Fig B. Jurkat cells treated with camptothecin. Cells were labeled with FAM-VAD-FMK for 1 hour. Caspase activity was detected using flow cytometry.

Citations

Granzyme B activates procaspase-3 which signals a mitochondrial amplification loop for maximal apoptosis - Sunil S. Metkar, Baikun Wang, Michelle L. Ebbs,Jin H. Kim,Yong J. Lee, Srikumar M. Raja, Christopher J. Froelich - Journal of Cell Biology, Volume 160, Number 6 2003; 875-885

Small-molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of myeloid Leukemia cells - Carter, Gronda, Wang, et.al - Blood, Vol 105, No 10 May 2005; 4043-4050

Fenretinide enhances rituximab-induced cytotoxicity against B-cell lymphoma xenografts through a caspase dependant mechanism - Gopal, Pagel, et.al - Blood, Vol 103, No 9 May 2004; 3516-3520

Anoxia Is Necessary for Tumor Cell Toxicity Caused by a Low-Oxygen Environment - Papandreou, Krishna, Kaper, et.al - Cancer Research 65 April 15, 2005; 3171-3178

IFN-y Induces Apoptosis in Ovarian Cancer Cells in Vivo and in Vitro - Wall, Burke, Barton, Smyth, Balkwill - Clinical Cancer Research Vol. 9 July 2003; 2487-2496

The removal of extracellular calcium: a novel mechanism underlying the recruitment of N-methyl-d-aspartate (NMDA) receptors in neurotoxicity - Xin, Zhao, Gang, et.al - European Journal of Neuroscience, Vol 21, Issue 3 Feb 2005; pp 622

Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death - Jagan R Muppidi & Richard M Siegel - Nature Immunology 5 2004; 182-189

Attenuation of apoptosis in enterocytes by blocking potassium channels - Levitan, Makhina, et. al - Am J Physiol Gastrointest Liver Physiol July 14, 2005

Induction of Apoptosis Using Inhibitors of Lysophosphatidic Acid Acyltransferase-ß and Anti-CD20 Monoclonal Antibodies for Treatment of Human Non-Hodgkin's Lymphomas - John M. Pagel, Christian Laugen, Lynn Bonham, Robert C. Hackman, et.al - Clinical Cancer Research Vol. 11 July 1, 2005; 4857-4866

Inhibition of PI3K, mTOR and MEK signaling pathways promotes rapid apoptosis in B-lineage ALL in the presence of stromal cell support - Bertrand, Spengeman, Shelton, et.al - Leukemia 2005; 19, 98-102

References

  • Slee, E. A., C. Adrain, and S. J. Martin. 1999. Serial Killers: ordering caspase activation events in apoptosis. Cell Death and Differ. 6:1067-1074.
  • 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.
  • Carcia-Calvo, M., E. Peterson, B. Leiting, R. Ruel, D. Nicholson and N. Thornberry. 1998. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273: 32608-32613.
  • Hirata, H., A. Takahashi, S. Kobayashi, S. Yonehara, H. Sawai, T. Okazaki, K. Yamamoto and M. Sasada. 1998. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. 187: 587-600.

Kit contents and Long Term storage

ReagentPart#Temperature
10X Wash BufferPart# 30262-8C
10X FixativePart# 30272-8C
Propidium IodidePart# 40132-8C
Lyophilized FAM Labeled Peptide InhibitorRefer to Product Datasheet2-8C
x