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Apoptosis Probes





IQFCS
  has optimized fluorescently labeled amino acid probes to quantitate apoptosis via active caspases in whole, living cells. The apoptotic probes do not use antibodies for detection. Instead, it uses an inhibitor sequence of caspases (such as VAD) linked to a green (carboxyfluorescein, FAM) or red (sulforhodamine, SR) fluorescent probe for easy detection. Fluorescently labeled apoptotic probes are cell-permeant eliminating the need to permeabilize the cell membranes.  If an active caspase enzyme is inside the cell, the caspase will form a covalent bond with the probe causing it to be trapped in the cell with a fluorescent signal for easy detection. Because the caspase enzyme itself binds with the probe, there is no interference from pro-caspases nor inactive forms of the enzyme. Importantly, with the addition of our necrotic stain, it is easy to distinguish apoptotic cells from necrotic cells see Figure 1.

 

ApPistaining.jpg
Figure 1. Distinction between apoptosis and necrosis using cell-permeant caspase probes.

AnnexVsProbes2.jpg

Cells were dual stained with the poly-caspase probe and Annexin. Figure 2 demonstrates that the caspase probes were significantly more specific than Annexin. A larger percentage of cells undergoing apoptosis were positive using the cell permeable probes. In contrast, Annexin failed to detect a significant percentage cells undergoing apoptosis.

In addition, International Qualex Flow Cytometry Systems optimized the cytotoxicity to distinguish apoptosis from necrosis resulting in the most sensitive cytolytic assay available. The total cytotoxicity assay not only is sensitive, it allows researchers to determine mechanisms involved in cell killing.

Apoptosis Presentation

In Vivo Apoptosis Detection

Apoptosis probes for Flow Cytometry - FAM conjugated pVAD

PRODUCT NUMBER
CASPASE
TEST SIZE
*Poly-caspase
125
CpVAD250
*Poly-Caspase
250
CpDEVD125
Caspases 3&7
125
CpDEVD250
Caspases 3&7
250
CpVDVAD125
Caspase 2
125

CpVDVAD250

Caspase 2

250

CpVEID125

Caspase 6

125

CpVEID250

Caspase 6

250

CpLEHD125

Caspase 8

125

CpLEHD250

Caspase 8

250

CpAEVD125

Caspase 10

125

CpAEVD250

Caspase 10

250

CpLEED125

Caspase 13

125

CpLEED250

Caspase 13

250

For fluorescent plate readers or microscope applications, please inquire within

 

References

1.Amstad, P. A., G. Yu, G.L. Johnson, B.W. Lee, S. Dhawan and D.J. Phelps. 2001. Detection of caspase activation in situ by fluo- rochrome-labeled caspase inhibitors. Biotechniques vol 31:608-614.

2. Bedner, E., P. Smolewski, P. Amstad, and Z. Darzynkiewicz. 2000. Activation of caspases measured in situ by binding of fluorochrome- labeled inhibitors of caspases (FLICA): correlation with DNA frag- mentation. Exp Cell Res vol 259:308-313
3. Buller, G., Bradford, J., Rothe, A., Janes, M., Salisbury, J., Ignatius, M., and Godfrey, W. 2004. Detection of apoptosis markers over time after induction. Poster presented at the American Society for Cell Biology,December,2004.
4. Darzynkiewicz, Z., E. Bedner,P.Smolewski, B.W.Lee and G.L. Johnson. 2001. Detection of caspases activation in situ by fluo- rochrome-labeled inhibitors of caspase (FLICA). Meth. Mol. Biol. vol 203:289-299.
5. Darzynkiewicz, Z., J. Grabarek, H.D. Halicka, and P.Smolewski. 2002. Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apopto- sis. Journal of Immunological Methods vol 265:111-121.
6. Gougeon, M. L., Lecoeur,H., 2002. Evaluation of apoptosis. Journal of Immunological Methods vol. 265, 1-2, 1 July 2002, pp. 1-2.
7. Grabarek, J., P.Amstad, and Z. Darzynkiewicz. 2002. Use of fluo- rescently labeled caspase inhibitors as affinity labels to detect activat- edcaspases. Human Cell vol 15(1):1-12.
8. Grabarek, J., Darzynkiewicz, Z. 2002. In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors. Exp. Hematology vol 30:982-989.
9. Graberek, J., Dragan, M., Lee, B. W., Johnson, G. L., Darzynkiewicz, Z. 2002. Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor. Int. J. Oncol vol 20:225-233.
10. Graberek, J., L. Du, G.L. Johnson, B.W. Lee, D.J. Phelps and Z. Darzynkiewicz. 2002. Sequential activation of Caspases and serine proteases (serpases) during apoptosis. Cell Cycle vol 1:124-131.
11. Pozarowski, P., X. Huang, D. H. Halicka, B. Lee, G. Johnson, and Z. Darzynkiewicz. 2003. Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: a caution in data interpretation. Cytometry vol. 55A:50-60.
12. Smolewski, P., E. Bedner, L. Du, T.C. Hsieh, J.M. Wu, D.J. Phelps, and Z. Darzynkiewicz. 2001. Detection of caspases activation by flu- orochrome-labeled inhibitors: Multiparameter analysis by laser scan- ning cytometry. Cytometry vol 44:73-82.
13. Smolewski, P., Grabarek, J., Halicka, H.D., and Darzynkiewicz, Z. 2002. Assay of caspase activation in situ combined with probing plasma membrane integrity to detect three distinct stages of apopto- sis. Journal of Immunological Methods vol 265:111-121.
14. Smolewski, P., Grabarek, J., Lee, B. W., Johnson, G. L., Darzynkiewicz, Z. 2002. Kinetics of HL-60 cell entry to apoptosis during treatment with TNF-alpha or camptothecin assayed by the stathmo-apoptosis method. Cytometry vol. 47:143-149.
15. Teodori, L., Grabarek, J., Smolewski, P., Ghibelli, L., Bergamaschi, A., De Nicola, M., and Darzynkiewicz, Z. 2002. Exposure of cells to static magnetic field accelerates loss of integrity of plasma membrane during apoptosis. Cytometryvol 49(3):113-118.
16. Valero, A., Merino, F., Wolbers, F., Luttge, R.Vermes, I., Andersson, H.and van den Berg, A.2005. Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device. Lab Chip vol 5:49-55.
17. Widen, R., C.A.Newton, T.W.Klein, and H.Friedman. 2001. Apoptosis of thioglycollate-elicited macrophages from A/J mice infected with virulent Legionella pneumophila. Abstracts of the General Meeting of the American Society for Microbiology vol 101:295-296.
18. Wlodkowic, D., Skommer, J., Pelkonen, J. 2006. Multiparametric analysis of HA14-1-induced apoptosis in follicular lymphoma cells. Leuk Res vol 30:1187-1192.
19. Wolbers, F., Buijtenhuijs, P., Haanen, C., and Vermes, I. 2004. Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used. Apoptosis vol 9:385-392.

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