Cell Apoptosis Assay

Updated: Jan 2

Apoptosis is a term referring to a program cell death. Although the meaning itself may evoke unpleasant feelings, this is one of the necessary processes the cells in all living organisms go through. This programmed cell death is a homeostatic mechanism to maintain cell populations in tissues and also a defense mechanism involved in immune reaction. [1]

Introduction of Cell Apoptosis

This programmed cell death are known to be predictable – of which occurs in predictable body parts such as tadpole tail loss as it grows into a frog, at predictable period such as formation of digits through interdigital cell death after certain period of growth, and even predictable in organism due to predetermined developmental plan. Not only in the growth, but also in adulthood, apoptosis continues to play an important role to maintain a balance with cell division and control the size of tissue, organ and body, and furthermore, eliminating cells causing negative effect such as damaged and transformed cells or those infected with pathogens. [1]

Apoptosis does not always work the same

Interestingly, apoptosis can be triggered physiologically and pathologically, and this triggered apoptosis does not have the same effect on all cells such that some cells react differently than others.

Physiological apoptosis is more of an overtime process involved in forming organs and tissues to maintain homeostasis whereas pathological apoptosis is involved in response to disease or injury occurred from stress or other conditions affecting negatively in cell survival. To distinguish clearly, physiological apoptosis is a predetermined and normal cellular process while pathological apoptosis is referred to as a response to abnormal conditions.

Process of Apoptosis

Now we get the meaning and the purpose of apoptosis, we could go in deeper level in finding what an apoptotic cell goes through. Getting the idea of how cell is programmed to be eliminated would help you appreciate its existence.

The process of apoptosis is as follows [3]:

1. Membrane blebbing

2. Cell shrinkage

3. Condensation of chromatin

4. Fragmentation of DNA

5. Rapid engulfment of the dead cells

How is apoptosis analyzed?

Apoptosis can be quantified using fluorophores such as Annexin V and DAPI reagent when applied to the cells. When using a flow cytometer or similar devices such as ADAMII LS (NanoEntek), early and late apoptosis are also detectable.

Figure 1. Dot plot showing apoptosis at different phases

This dot plot shows the difference between apoptosis and necrosis including early and late apoptosis at different areas with different distribution (from the bottom left, each area represents viable cell, early apoptosis, late apoptosis, and necrosis, respectively, in counterclockwise). In the dot plot of the group of cells treated with camtothecin (CPT), an anticancer drug inducing apoptosis, late apoptosis proportion is approximately 45%, whereas the untreated group only shows about 2% of late apoptosis.

The process of apoptosis can also be observed via live-cell imaging. In industries like pharmaceuticals, where researching the effect of chemical substances to discover new drugs to treat cancer, etc., is vital, it is crucial to observe and analyze the entire process of cell response to specific treatment. In the video above cell apoptosis process can be observed by comparing with the control group, the experimental group treated with camptothecin (CPT), an anticancer drug inducing apoptosis. Over time, the experimental group treated with CPT shows dead cells that express green fluorescent light (on right black screen), whereas the control group does not show any.

To sum up, apoptosis is a program cell death which can be quantified and analyzed using different assays. Depending on how cells are triggered, cell death can occur in two forms – apoptosis and necrosis. To quantify each phase of cell death, dot plot based would be used and to observe how cells die over time, live cell imaging would be required.

References:

1. Elmore, Susan. “Apoptosis: A Review of Programmed Cell Death.” Toxicologic Pathology, U.S. National Library of Medicine, June 2007, www.ncbi.nlm.nih.gov/pmc/articles/PMC2117903/.

2. https://www.sciencedirect.com/sdfe/pdf/download/eid/3-s2.0-B9780123704580500153/first-page-pdf

3. Renehan, A G, et al. “What Is Apoptosis, and Why Is It Important?” BMJ (Clinical Research Ed.), U.S. National Library of Medicine, 23 June 2001, www.ncbi.nlm.nih.gov/pmc/articles/PMC1120576/#:~:text=Apoptosis%20describes%20the%20orchestrated%20collapse,the%20corpse%20by%20neighbouring%20cells.