Nuclear condensation, DNA fragmentation and membrane disruption during apoptosis

Chromatin condensation and genomic DNA fragmentation, together with cell membrane blebbing, are considered morphological hallmarks of the terminal stages of apoptosis. 

Chromatin condensation

​During apoptosis, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert, highly condensed form. When stained with DNA-binding nuclear dyes, the compacted chromatin will be brighter than the chromatin from non-apoptotic cells, and the condensed nuclei can be easily identified by fluorescence microscopy (qualitative detection) and/or flow cytometry (quantitative detection).

ab139479 nuclear condensation assay kit

​​​​Nuclear Condensation Assay Kit (ab139479):  Flow cytometry analysis of untreated staurosporine-treated Jurkat cells.  The raw data shown in panels A and B was used for gating cell populations (rectangles) for analysis.  Panels C and D represent the separation of healthy and apoptotic nuclei based on their characteristic fluorescence.

Genomic DNA fragmentation

Condensed chromatin can be fragmented by a specific nuclease called Caspase-Activated DNase (CAD). Activation of CAD by the caspase cascade leads to specific cleavage of the DNA at the internucleosomal linker sites between the nucleosomes, generating fragments of ~ 200 base pairs known as DNA ladders.

​​​​A classical method to detect DNA ladders is to examine fragmented genomic DNA on a agarose gel - view our Apoptosis DNA fragmentation protocol  here. This is a simple, semi-quantitative method that provides a robust answer. Some tips to help you get the best from the experiment are:

  • The DNA in the sample will make it very viscous and sticky. Use the DNA sample loading buffer at a higher concentration than you would normally to ensure the sample does not float away from the well (for example, use 10 µL 10X loading buffer in 50 µL to compensate for the higher viscosity.
  • Prepare a gel with 1.8 - 2% agarose. The high agarose concentration provides the necessary resolution to see the steps in the ladder.
  • Run the gel at lower voltage for a longer time than you would normally to over heating and subsequent deformation of the bands.

​An alternative method for detecting DNA fragmentation involves the identification of nicks (or strand breaks) using a TUNEL assay. The TdT dUTP nick end labeling (TUNEL) technique is based on the ability of erminal deoxynucleotidyl transferase (TdT) to end-label nicks in the DNA caused by CAD. TdT has the ability to label 3' blunt ends of double stranded DNA with deoxyuridine, independently of the template.

Increase of subG1 population

An increase in the percentage of cells stalled at G1 is another consequence of DNA fragmentation, and can be readily detected with a flow cytometer. Once cells are permeabilized with, for example, a 70% ethanol solution, the DNA fragments will leak out and create a population of cells with reduced DNA content. When cells are stained with a DNA staining dye such as PI, a DNA profile representing cells in the different stages of the cell cycle (G1, S-phase and G2M) can be observed by flow cytometry. The apoptotic cells are easily identified as the subG1 population seen to the left of the G1 peak. The subG1 fraction will include all the dead cells in the population regardless of the type of cell and therefore this parameter on its own is not a good indicator of apoptosis.

ab139418 propidium iodide flow cytometry kit

Healthy population of HeLa cells analyzed with Propidium Iodide Flow Cytometry Kit (ab139418):  Flow cytometer software was used to establish markers on a histogram plot to quantify the percentage of cells with <2N (dead cells), 2N (G1 phase), 2N-4N (S-phase), 4N (G2 phase) and >4N (M phase) content.

Nuclear cell membrane blebbing

Together with DNA fragmentation, the final execution phase of apoptosis is characterized by dynamic membrane blebbing and cell contraction. During apoptosis, the cell cytoskeleton breaks up, causing some parts of the cell membrane to bulge outwards. The bulges eventually separate from the cell taking a portion of the cytoplasm with them and forming what are known as apoptotic bodies.

Membrane blebbing can be observed in live cells using phase-contrast microscopy. If you are not able to use live cells or would like to use cells that you have prepared for studying other parameters (for example, cells harvested for DNA fragmentation quantification or chromatin condensation), you can detect caspase substrates associated with apoptotic membrane blebbing. Be aware, however, that this is an indirect method and it may give you false positive/negative results.

Caspase substrates associated with apoptotic membrane blebbing include: