Flow Cytometry and Small Particles Detection (FCSPD) Facility

Apoptosis

Apoptosis is a genetic process by which a cell is programmed to die. This process is critical for development and proper function of an organism. Two main pathways lead to Apoptosis (right figure; Wlodkowic D, Met Cell Biol. 103, 2011): Extrinsic (activated mainly by death ligands, such TNFR, TRAIL, and FasL) and Intrinsic (activated by DNA damage, hypoxia, survival factor deprivation, etc.), resulting in the activation of a family of cysteine proteases, caspases. As a consequence of caspase activation a proteolytic cascade occurs leading to cell death and elimination of dying cells.

Morphological (cell volume fluctuations, phosphatidylserine “flipping”, loss of membrane permeability, chromatin condensation) and biochemical (mitochondrial potential changes, caspase, other signaling events) changes are characteristic of Apoptosis. Importantly, most of those changes can be detected and measured by flow cytometry (bottom fig; Darzynkiewicz Z, Cytometry 27, 1997).

However, depending on the cell type and its stage of differentiation/activation, the apoptotic process may have a lot of phenotypic variations. Then, to properly characterize Apoptosis always measure it using several different methods, preferably in the same sample. Multiparametric flow cytometry is ideal for this. Do not only measure cell death, but characterize it! (advice from Dr. Bill Telford, NCI Flow Cytometry Core Lab).

Measuring Cell Death by Flow Cytometry

Changes in Forward and/or Side Scatter (FSC/SSC): a measure of cell shrinkage and cytoskeleton collapse (figure).

DNA binding dyes: a measure of chromatin condensation and DNA fragmentation (figure; see Viability protocols).

Annexin V binding: a measure of phosphatidylserine (PS) flipping. One of the earliest events during apoptosis is the translocation of PS from the inner to outer face of the plasma membrane (figure).

DNA fragmentation by TUNEL assay: During apoptosis there is a presence of nicks in the DNA (DNA fragmentation) that can be identified by terminal deoxynucleotidyl transferase (TdT), an enzyme that catalize the addition of dUTP (secondarily labeled with a fluorochrome) (figure).

Active Caspase detection: Caspases (cystein-dependent, aspartate-specific proteases) are activated during apoptosis and are integral to cell death. Caspase "initiators" (caspases 2, 8, 10) can initiate the apoptotic cascade. Caspase "effectors" (caspases 3, 6, 7) perform proteolytic events leading directly to cellular breakdown. Increase caspase active alone does not necessarily indicate apoptosis. Look for caspase activation together with other markers of cell death!