DNA Fragmentation Assay

For Late Apoptosis Evaluation Using the NucleoCounter® NC-3000™

  • Easily measure DNA fragmentation levels
  • Acquisition and analysis in one simple step
  • User-friendly protocol with predefined settings
  • No RNase treatment required
  • No calibration required
  • Clear data presentation with Plot Manager feature
  • Automated PDF reports
  • Export data in FCS/ACS formats

Overview of the DNA Fragmentation Assay

During apoptosis, proteins act on chromosomal DNA to fragment the DNA into ladders. This late-stage apoptotic event is detected by measuring DNA content in a way similar to that done in our Cell Cycle Assay. In apoptotic cells you will see a dissolution of peaks at the G1 and G2 phases, and the rise of a “sub G1 peak” which grows with the progression of the chromosomal DNA laddering.

The mechanism of the DNA degradation is the activation of calcium- and magnesium-dependent nucleases as apoptosis progresses. As a result, within the DNA there are nicks and double-strand breaks causing fragmentation1. This is a late-stage apoptotic event, which can be detected by quantifying the cells’ DNA content. Late-stage apoptotic cells will have less DNA content and can thereby be identified as being in the sub-G1 phase.

Assay Principle

Using fluorescence microscopy and image analysis, the NucleoCounter® NC-3000™ automatically detects cells with fragmented DNA (sub-G1 cells).

After staining fixed cells with DAPI, the sample is analyzed using the NucleoCounter® NC-3000™ system, where cellular fluorescence is quantified and apoptotic cells with fragmented DNA are identified as a sub-G1 peak in a DNA content histogram displayed on the computer screen.

Markers in the histogram can be used to demarcate apoptotic cells.

Results Presented in Plot Manager

Jurkat cells were grown in the absence (upper row) or in the presence (lower row) of camptothecin and cells were analyzed using the DNA Fragmentation Assay and a NucleoCounter® NC-3000™.

Scatter plots and histograms were obtained using the NucleoView™ NC-3000™ software. Markers in the displayed histograms were used to demarcate cells with fragmented DNA (sub-G1 cells). The colored histograms are a merge of untreated (blue line) and camptothecin treated (red line) samples.

References

  1. BD Larsen and CS Sørensen: The caspase‐activated DNase: apoptosis and beyond. FEBS J. 2017; 284(8): 1160-1170.

Publications

Cytotoxic and proapoptotic effect of doxycycline – An in vitro study on the human skin melanoma cells.

  • Rok J, Karkoszka M, Rzepka Z, et al. ,

  • Toxicology in vitro_ an international journal published in association with BIBRA ,

  • 2020,

  • 65:104790

  • Instrument: NucleoCounter® NC-3000™
  • Cell Type: Cancer Cells
  • Research area: Small Molecule Drugs

Doxycycline is a semisynthetic, second generation tetracycline. Currently, it is used, among others, in the treatment of acne and skin infections. Moreover, doxycycline has many valuable nonantibiotic properties, including anti-inflammatory, immunosuppressive and anticancer effects. Recent studies showed that the drug...

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