• Much more Reliable as compared to TUNEL assays - No False positive signals
• Multiple parameter detection of DNA damage and caspase 3 activity simultaneously
• Readout - Flow cytometry, Fluorescence 96 well plate reader, Fluorescence microscope
• Yields both quantitative and qualitative results. Gives a strong positive signal.
• Ease Of Use: Dual parameter results to confirm apoptosis in cells./li>
• Compatible with human, mouse, rat, bovine, porcine species/li>

1. Antibody to Single Stranded DNA
A widely used cytochemical technique for evaluation of DNA damage associated with apoptosis is the terminal deoxynucleotidyl transferase-mediated in situ end labeling or TUNEL assay. However the TUNEL assay has its drawbacks in that false positive staining makes the assay unreliable as a marker for apoptosis 1-5. A more universal and specific marker for detecting apoptosis associated DNA damage is to measure the morphological changes in nuclei that reflect chromatin condensation into compact masses. 6-7. Further biochemical and cytochemical studies have demonstrated the increased susceptibility of apoptotic DNA to thermal denaturation. Analysis of nuclei by scanning calorimetry to detect thermal induced DNA denaturation and analysis of DNA fragmentation by electrophoresis have shown that intact apoptotic DNA is susceptible to denaturation at lower temperatures then that of non-apoptotic cells 8.

2. Antibody to Active Caspase 3
Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes participate in a series of reactions that are triggered in response to pro-apoptotic signals and result in cleavage of protein substrates, causing the disassembly of the cell 13. Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large (~20 kD) and two small (~10 kD) subunits to form two heterodimers which associate in a tetramer 14-16. As is common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity 17. Caspase 3, also known as CPP-32, Apopain or Yama, is a key effector caspase in the apoptotic pathway 18. It is present in many different cell lineages and is responsible for the cleavage of a variety of molecules such as poly ADP-ribose polymerase (PARP), protein kinase Cd, actin and DNA-dependent protein kinase 19-20.

Cell Technology introduces a dual parameter antibody based assay to detect DNA damage (heat denatured single stranded DNA: ssDNA)9-12 and active caspase 3 in apoptotic cells. The assay utilizes a monoclonal antibody generated against ssDNA and a primary rabbit affinity purified polyclonal antibody raised against amino acid 163-175 of murine caspase 3 12. This neo epitope is present on the p18 subunit of cleaved caspase 3 21.

DNA Damage detection: Excitation: 488nm, Emission: 530nm (FL1)
Caspase 3 detection: Excitation: 633nm, Emission: 670nm (LP) (FL4)

1. Charriaut–Marlangue C, Ben-Ari J (1995) A cautionary note on the use of TUNEL to determine apoptosis. NeuroReport 7:61–64
2. Grasl–Kraipp B, Ruttkau–Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte–Hermann R (1995) In situ detection of frag-mented DNA (TUNEL) fails to discriminate among apoptosis, necrosis and autolytic cell death: a cautionary note. Hepatology 21:1465–146
3. Didenko VV, Hornsby PJ (1996) Presence of double-stranded DNA breaks with single-base 39 overhangs in cells undergoing apopto-sis but not necrosis. J Cell Biol 135:1369–1376
4. Ohno M, Takemura G, Ohno A, Misao J, Hayakawa Y, Minatogu-chi S, Fujiwara T, Fujiwara H (1998) “Apoptotic” myocytes in infarct area in rabbit hearts may be oncotic myocytes with DNA fragmentation: analysis by immunogold electron microscopy combined with in situ nick end-labeling. Circulation 98:1422–1430.
5. Stadelmann C, Bruck W, Bancher C, Jellinger K, Lassmann H (1998) Alzheimer disease: DNA fragmentation indicates in-creased neuronal vulnerability, but not apoptosis. Neuropathol Exp Neurol 57:456–464
6. Willingham MC (1999) Cytochemical methods for the detection of apoptosis. J Histochem Cytochem 47:1101–1109
7. Zamzani N, Kroemer J (1999) Condensed matter in cell death. Nature 401:127–128.
8. Allera C, Lazzarini G, Patrone E, Alberti I, Barboro P, Sanna P, Melchiori, A, Parodi S, Balbi C (1997) Condensation of chromatin in apoptotic thymocytes shows a specific structural change. J. Biol Chem 272:10817–10822.
9. Frankfurt OS (1990) Decreased DNA stability in cells treated with alkylating agents. Exp Cell Res 191:181–185.
10. Frankfurt OS, Robb JA, Sugarbaker EV, Villa L (1996) Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp Cell Res 226:387–397.
11. Frankfurt OS (1994) Detection of apoptosis in leukemic and breast cancer cells with monoclonal antibody to single-stranded DNA. Anticancer Res 14:1861–1870.
12. Oskar S. Frankfurt and Awtar Krishan (2001). Identification of Apoptotic Cells by Formamide-induced DNA Denaturation in Condensed Chromatin. The Journal of Histochemistry & Cytochemistry Volume 49(3): 369–378, 2001
13. 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.
14. 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.
15. 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.
16. 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.
17. Kumar, S. 1999. Mechanisms mediating caspase activation in cell death. Cell Death and Differ. 6: 1060-1066.
18. Alnemri, E.S. et al (1996) Cell 87:171
19. Trends Biochem Sci 22,388 (1997)
20. Nature 376, 37 (1995).
21. Srinivasan A, et al Cell Death and Differentiation (1998) 5: 1004-1016