Apo ss DNA

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Apo ssDNA
An antibody assay to detect DNA Damage (Single Stranded) in apoptotic cells

Key Benefits:

Much more Reliable as compared to TUNEL assays - No False positive signals
Readout - Flow cytometry, 96 well plate reader, Fluorescence microscope .
Yields both quantitative and qualitative results. Gives a strong positive signal.
Ease Of Use: Results in less than 60 minutes
No need to run DNA ladder assays to detect DNA damage in cells

Introduction:

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 (see figure 1) ^1-5. A more universal and specific marker for apoptosis is the morphological changes in nuclei that reflect chromatin condensation in to 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.

Assay Principle:

Cell Technology introduces Apo ssDNA, an antibody based assay to detect DNA damage (single stranded DNA: ssDNA) in Apoptotic Cells.

The assay utilizes an antibody generated against ssDNA. This antibody recognizes large stretches of heat-denatured ssDNA only in apoptotic cells ^9-12

References:

1. Charriaut–Marlangue C, Ben-Ari J (1995) A cautionary note on the use of TUNEL to derermine 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 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

Kit contents and Long Term storage:

1. Mouse anti single stranded DNA………………… Part # 1003 -70°C aliquot

2. Anti mouse IgM FITC labeled…………………… Part # 2005 -20°C

3. Block Buffer…………………………..…………………………… Part # 3033 4-8°C

4. 10 X Wash Buffer…………………………………………… Part # 3026 4-8°C

The following kits are available:
Catalog #	Size*	Price
APODNA-1	25 Tests	€225
APODNA-2	100 Tests	€745

(A)

(B)

Fig (A). MAb to ssDNA staining of apoptotic but not of necrotic cells. Fluorescence distributions of MDA-468 cells heated in formamide and stained with MAb F7-26 were generated on a flow cytometer. Apoptosis was induced by staurosporine and necrosis was induced by sodium azide, saponin, or hyperthermia. Note that apoptotic cells are intensely stained with the MAb, whereas the fluorescence profiles of necrotic and control cells are similar.

Fig (B). TUNEL staining of apoptotic and necrotic cells. Fluorescence distributions of MDA-468 cells stained with TUNEL were generated on a flow cytometer. Apoptosis was induced by staurosporine and necrosis was induced by sodium azide, saponin, or hyperthermia. Note that cells at early stage of apoptosis (staurosporine 3 hr) are weakly stained, whereas late apoptotic cells (staurosporine 5 hr) and necrotic cells are intensely stained by TUNEL ¹².