Colourful death: Six-parameter classification of cell death by flow cytometry—Dead cells tell tales

Abstract

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

Keywords::

• Apoptosis
• necrosis
• phosphatidylserine
• mitochondrial membrane potential
• membrane integrity

Acknowledgement

This work was supported by SFB643 TP B5, by the Emerging Fields Initiative (EFI) of the FAU Erlangen-Nürnberg, by the K & R Wucherpfennig-Stiftung, by the Interdisciplinary Center of Clinical Research (IZKF) of the University Hospital Erlangen-Nürnberg and by training Grants GK SFB 643 and GK1660 from the German Research Society (DFG).

Declaration of interest : The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.