How to prepare samples for DNA fragmentation in NC3000

Background

During apoptosis, calcium- and magnesium-dependent nucleases are activated which degrade DNA. This means that within the DNA there are nicks and double-strand breaks causing fragmentation. This late event of apoptosis is detected using DNA content of mammalian cells in order to detect apoptotic cells with fragmented DNA (sub-G1 cells). The DNA fragmentation assay in the Nucleocounter NC3000 enables the quantification of the DNA content.

Below is described how to prepare samples for DNA fragmentation assay.

Materials necessary for DAPI staining 

– Cells to be stained

– Phosphate buffered saline (PBS)

– Fixative: 70% ethanol

– Solution 3 (1 µg/ml DAPI, 0.1% triton X-100 in PBS)

– NC-Slide A2™ or NC-Slide A8™

Always include an un-treated negative control for reference. It is also important to have an equal cell concentration in treated vs. untreated samples to avoid artificial changes in the peak of the histogram. It is recommended to count the cells prior to fixation.

For a more detailed description of the protocol 

Protocol for staining of cells 

  • Fixation of cells in ethanol – important that the cells are kept in the fixative for at least 12 hours before performing assay.
  1. For adherent cells. Harvest cells by trypsinization and pool the trypsinized cells with cells floating in the medium (latter consist of detached mitotic, apoptotic and dead cells).  

For both suspension and adherent cells centrifuge the samples 5 min. at 500 g at room temperature (RT). Wash once with PBS. Count cells and thoroughly resuspend 1×10^6 to 2×10^6 cells in 0.5 ml PBS.

Important note: For proper staining it is crucial to keep the cell density within the range of 2×10^6 to 4×10^6 cells/ml. In case of limited amounts of cells, the procedure can be scaled down, e.g. use 2×10^5 to 4×10^5 cells in 0.1 ml PBS in step 1.

  • Add 4.5 ml of 70% ethanol to each of an appropriate number of 10-15 ml tubes. Keep on ice.
  • Transfer the cell suspensions (prepared in step 1) into appropriate tubes containing ice-cold 70% ethanol, vortex rigorously, and keep the cells in the fixative for at least 12 hours.

Important note: it is essential to have a single-cell suspension at the time that cells are mixed with ethanol. Cells can be stored in 70% ethanol for several weeks at 0-4°C.

  • Before assaying the samples wash the cells in PBS.
  • Centrifuge ethanol-suspended cells for 5 min. at 500 g. Decant ethanol thoroughly.

Note: Cell pellet may be loose. Make sure that no cells are lost in this and subsequent washing steps.

  • Suspend cell pellet in 5 ml PBS, leave for 50 sec, and centrifuge 5 min. at 500 g.
  • Staining of cells with DAPI (Solution 3)
  • Resuspend cell pellet in 0.5 ml Solution 3 and incubate for 5 minutes at 37°C.
  • Turn on the NucleoCounter® NC-3000™ by starting the NucleoView™ NC-3000 software.
  • Load the samples in either NC-Slide A2™ (~30 µl per chamber) or NC-Slide A8™ (~10 µl per chamber).
  • Place the loaded slide on the tray of the NucleoCounter® NC-3000™. In the NucleoView™ NC-3000 software click on the folder button, a Select Protocol window opens up. 

In Organism section choose Mammalian, in Analysis section choose Apoptosis and in Protocol section choose DNA Fragmentation Assay NC-Slide A2 or DNA Fragmentation Assay NC-Slide A8 and press Select. Add in OperatorSample ID and press RUN.

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