Apoptosis Assays by Flow Cytometry

Apoptosis, also known as programmed cell death, is a normal process of the development and health of multicellular organisms. In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis is a controlled, regulated process and confers advantages during an organism's life cycle. A series of changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation have occured during apoptosis. Apoptosis functions in multiple contexts. In development, apoptosis allows for the definition of shape. For example, the fingers of fetal hands are webbed, but the tissue between the fingers separates as the cells die. It is also beneficial as a natural anti-cancer mechanism. When a cell's DNA becomes damaged, a cell can undergo apoptosis, preserving the healthy state of the organism. Cells that have become irreparably damaged due to disease also undergo apoptosis.

The following features in apoptosis can be observed using flow cytometry.

Cell shrinkage

Forward scatter (FSC) is the light from the illuminating beam that has been bent at a small angle as it passes through a cell, which is relevant to the size of cells. Side scatter (SSC) Light that bounces off particles and is deflected 90 degrees, which is relevant to the refractive index of plasma membrane and intracellular structure. When apoptosis occurs, cell shrinkage, pycnosis and increasing content of particles happens at the same time. So decreasing of FSC and increasing of SSC can be observed by flow cytometry. While in necrosis cells, FSC and SSC both increasing.

Changes of plasma membrane

In early apoptosis, phosphatidyl serine (PS) residues, which is positioned inside the plasma membrane, expose to the cells outer surface. Annexin V is human vascular anticoagulant and with high affinity for PS. Annexin V-FITC/PI staining method is widely used in the the detection of early apoptosis.

Changes of DNA content

At a late stage in the apoptosis, endonucleases break the linkers between the nucleosomes, one of the units of chromatin organisation. Consequently, large numbers of small fragments of DNA, whose sizes are oligomers of about 180 bp, accumulate in the cell. If cells are fixed in ethanol and subsequently rehydrated, some of the lower molecular weight DNA leaches out, lowering the DNA content. These cells can be observed as a 'sub-G1' peak in a DNA histogram.

DNA degradation

The presence of nicks in the degraded DNA can be recognized by terminal deoxynucleotidyl transferase that then catalyzes the addition of dUTPs. dUTPs can be labelled by FITC to be visualized by flow cytometry. This methods, known as TUNEL, can effectively detect late apoptosis cells, However, it may also label cells under going necrosis or cells that have suffered severe DNA damage, so it should be carefully done to achieve the purpose of mainly labeling late apoptotic cells.

Activation of caspases

Caspases are a family of cysteine proteases that play essential roles in apoptosis. There are a number of caspases in mammalian cells that have been shown to be involved in the early stages of apoptosis, e.g. Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9 and Caspase 10. The functions of these enzymes are not yet entirely clear, but it appears that after an initial signal to the cell to undergo apoptosis, they may be responsible for the activation, amplification and execution of the apoptotic cascade.

Caspase can be detected in three ways:

• By detecting the active form of the enzyme using a specific antibody;
• By using a fluorochrome labelled inhibitor peptide that binds to the active site of the caspase;
• By using a non-fluorescent substrate for the enzyme which yields a fluorescent product if the enzyme is active.

Other changes

Other methods can also be used in the detection of apoptosis. For example, rhodamine 123 (Rh123) can detect the changes in the mitochondrial membrane potential. Acridine Orange (AO) can detect proton pump function of lysosome. ssDNA monoclonal antibody can recognize apoptosis cells specifically. Other apoptosis-related proteins, such as P53, C-myc, Fas antigen, TNF, bcl-2 family, cyclin and ras, can also be used in the detection of apoptosis.