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Apo ssDNA™

Apo ssDNA™ Cell Technology Caspase / Apoptosis Detection
Apo ssDNA™
An antibody assay to detect DNA Damage (Single Stranded) in apoptotic cells

$225.00$745.00

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Product code: APODNA
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Product Description

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

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.

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 cells9-12

apoptosis_induced_dna_damage2Fig (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.


apoptosis_induced_dna_damage1Fig (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.

Citations

Overexpression of human truncated peroxisome proliferator-activated receptor {alpha}induces apoptosis in HL-1 cardiomyocytes - Javier Beaumont Teresa Arias, Susana Ravassa and Javier Díez - Cardiovacular Research 2008 79(3):458-463

Inhibition of Src reduces gemcitabine-induced cytotoxicity in human pancreatic cancer cell lines - N. Ichihara, Y. Kubota, A. Kitanaka, T. Tanaka, T. Taminato - Cancer Letters Volume 260, Issue 1, Pages 155-162

Inhibition of Eyes Absent Homolog 4 expression induces malignant peripheral nerve sheath tumor necrosis - S J Miller , Z D Lan , A Hardiman , J Wu , J J Kordich , D M Patmore , R S Hegde , T P Cripe , J A Cancelas , M H Collins & N Ratner - Oncogene 29, 368–379 (21 January 2010) | doi:10.1038/onc.2009.360

HDAC inhibitors activate NF-κB in human leukemia cells through an ATM/nemo-related pathway - Roberto R. Rosato, Sarah S. Kolla, Stefanie K. Hock, Jorge A. Almenara, Ankita Patel, Sanjay Amin, Peter Atadja, Paul B. Fisher, Paul Dent, and Steven Grant - J. Biol. Chem. 2010 285: 10064-10077. First Published on January 11, 2010, doi:10.1074/jbc.M109.095208

Cytotoxic Effects of Bilberry Extract on MCF7-GFP-Tubulin Breast Cancer Cells - Vy Nguyen, Jessica Tang, Emin Oroudjev, Choong Jae Lee, Cecilia Marasigan, Leslie Wilson, George Ayoub - Journal of Medicinal Food April 2010, 13(2): 278-285

References

  • Charriaut–Marlangue C, Ben-Ari J (1995) A cautionary note on the use of TUNEL to derermine apoptosis. NeuroReport 7:61–64
  • 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
  • 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
  • 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.
  • 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
  • Willingham MC (1999) Cytochemical methods for the detection of apoptosis. J Histochem Cytochem 47:1101–1109
  • Zamzani N, Kroemer J (1999) Condensed matter in cell death. Nature 401:127–128.
  • 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.
  • Frankfurt OS (1990) Decreased DNA stability in cells treated with alkylating agents. Exp Cell Res 191:181–185.
  • 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.
  • Frankfurt OS (1994) Detection of apoptosis in leukemic and breast cancer cells with monoclonal antibody to single-stranded DNA. Anticancer Res 14:1861–1870.
  • 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

ReagentPart#Temperature
Mouse anti single stranded DNAPart # 1003-70°C aliquot
Anti mouse IgM FITC labeledPart # 2005-20°C
FixitivePart # 30334-8°C
10X Wash BufferPart # 30374-8°C
1X DNA Denaturing BufferPart # 30364-8°C
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