University of Dundee

Cell Death and Apoptosis

Membrane integrity

Possibly the simplest assay for cell death is measurement of plasma membrane integrity. This can be assessed in two ways: The ability of a cell to prevent a fluorescent dye from entering it and the ability of a cell to retain a fluorescent dye within it.

As a cell dies it's plasma membrane becomes permeable allowing fluorescent dyes present outside the cell to enter it and fluoresce. The most common dyes used for this purpose are dyes that label nucleic acids. These are available with a variety of excitation and emission spectra, allowing cell viability to be assessed in combination with many different fluorophores. The most commonly used dyes are DAPI, propidium iodide, 7AAD, and ToPro-3.

Other dyes, such as CFDA and Indo-1, are readily taken up by live cells, and upon internalisation are cleaved into a fluorescent form that can no longer cross the membrane. The result is that live cells become loaded with the dye, but dead cells, that do not have the enzymes necessary for cleavage of the non-fluorescent pre-cursor, will not. As a cell dies, the membrane becomes permeable and the fluorescent dye leaves the cell resulting in a loss of fluorescence.

AnnexinV

A well-established feature of apoptosis is the externalisation of the lipid phosphatidyl serine (PS) from the inner to the outer plasma membrane. Annexin-V (five) is a protein that specifically binds PS and fluorescent labelling of the annexin-V enables the flow cytometric detection of externalised PS, and hence apoptotic cells.

When used in conjunction with a live/dead cell discriminator that measures membrane integrity (such as PI, 7AAD, and DAPI), early apoptotic cells (annexin-V positive only) can be distinguished from late apoptotic/necrotic cells (annexin-V and PI/7AAD positive).

The early apoptotic phase can be quite rapid and can often be missed, making it appear that cells are either live or late apoptotic/necrotic. It is therefore often necessary to perform a time course experiment to prove that cells are in fact traversing through early apoptosis before reaching late apoptosis/necrosis.

While the annexin-V binding assay is fairly robust when used in suspension growing cells, it's use can be a little tricky when assessing apoptosis in adherent cells. The most common methods of cell detachment use trypsinisation and/or EDTA, both of which can seriously affect the assay. Trypsinisation can affect both membrane integrity and PS localisation and EDTA will sequester Ca2+, which is essential for the binding of annexin-V. It is therefore important to include the appropriate controls and optimise the assay for each cell type when using these methods of cell detachment.

Caspases

An early, biochemical event in the apoptotic process is the activation of the caspase cascade. Caspase activation involves the cleavage of the in-active caspase by enzymes (often other caspases) into an active form. This process can be measured in a number of ways and subsequently detected by flow cytometry.

The first technique uses a fluorescently labelled antibody specific for the caspase in its activated form, while showing minimal binding to in-active caspase. To allow intracellular staining it is necessary to both fix and permiablise the cells prior to labelling with the antibody. This has the advantage of allowing samples to be stored after the fixation step and before labelling, making it useful when performing time course experiments. The permiablisation of the cells also makes it easy to combine the assay with the measurement of DNA content to determine from which compartment of the cell cycle apoptosis is occurring.

Another method for the measurement of activated caspases is the use of fluorescently labelled inhibitors of caspases (FLICA). These are cell permeable peptides that bind strongly to the activated form of caspases, with different peptide sequences being specific for different caspases. The assay is performed on unfixed and unpermiablised cells and enables early apoptotic cells to be distinguished from late apoptotic/necrotic cells in their ability to exclude probes for membrane integrity, such as propidium iodide or 7-AAD.

The final method for the detection of activated caspases uses a cell permeable non-fluorescent substrate that is cleaved within the cell by active caspases into a fluorescent product. In this way apoptotic cells can be distinguished from non-apoptotic cells. Again, this assay is performed on unfixed and unpermiablised cells and may be combined with the assessment of membrane integrity to distinguish early apoptotic cells from late apoptotic/necrotic cells.