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Locating Your Cellular Apoptosis Squad: Annexin V Staining Assays

In real life, cells are instructed to commit suicide for the greater good of the organism. The programmed cell death (apoptosis) is important during development of a multi-cellular organism. A good example you will appreciate is the dis-appreance of the tail from a tadpole as it turns into a frog. On the reverse, the lack of cellular apoptosis leads to uncontrolled cell growth and various malignancies. Therefore, scientists are always searching for new ways to study cell death. Flow cytometry is an awesome invention that allows you to examine each cell’s surface protein expression and internal protein secretion. Flow cytometry revolutionized the field of immunology and continues to advance cell biology research. There are currently several different standard methods that scientists have developed to study cell death using flowcytometry.1 Today, I would like to introduce a relatively simple and reliable method to study cell death via detection of phospholipid moieties on the cell surface.

How does Annexin V staining work in an apoptosis assay?

Annexin V is a protein that binds to phosphotidylserine (PS) in a calcium-dependent manner. Since PS is only present on the cell surface when the cell is undergoing cell death, it is a useful regent to study apoptosis. By incorporating cell permeability dyes such as propidium iodide (PI) or 7-AAD, Annexin V staining via flow cytometry is a fast and reproducible way to look at different stages of cell death (see Fig 1). With proper gating on the cell populations, figure 1 shows a typical staining pattern on cells undergoing apoptosis using Annexin V and 7-AAD quadrant 1 (Q1) shows Annexin V and 7-AAD negative populations, which are healthy cells. Q2 shows Annexin V-positive and 7-AAD-negative populations, which are cells undergoing apoptosis. Finally, Q3 represents Annexin V and 7-AAD positive populations, which are at the terminal stage of cell death.

Annexin V Staining Example

Figure 1. Dot plot diagram from FACs showing progression of cell death. Q1. Double negative (Annexin V and 7-AAD negative) healthy cells. Q2. Annexin V positive, 7-AAD negative apoptotic cell. Q3. Annexin V & 7-AAD double positive necrotic cell

Procedure for Annexin V staining

Following is a typical procedure for Annexin V staining

Note: all the washing and incubation should be done in 1x binding buffer.2

  1. Harvest and wash cells in PBS
  2. Suspend cells in 1x Annexin V binding buffer that contains calcium
  3. Add the right concentration of flurochrome-conjugated Annexin V2
  4. Incubate for 15 min at room temp
  5. Wash twice with binding buffer
  6. Add PI or 7-ADD and incubate for 5 minutes
  7. Suspend the samples in Annexin V binding buffer and analyze as soon as possible

Some key points on using Annexin V staining to study cell death

1. Binding is reversible and requires a specific buffer

Due to its requirement of calcium for binding, the assay requires use of a Annexin V binding buffer, which is usually supplied when you purchase a kit. Note that the binding is reversible and the samples should be run as soon as possible to avoid the increase in background level. 

2. Choose the appropriate flurochrome for Annexin V.

When running multiple colors, which is usually the case with any assays nowadays, mix and match your flurochromes to avoid spectra over-lap and compensation problems

3. Cell permeability dyes (PI or 7-AAD) are required and fixation is not possible

Annexin V assay requires the incorporation of DNA-binding dyes (i.e., PI/7-AAD) to tell whether the cells are at the early (Annexin V positive, 7AAD negative) stage and actively undergoing apoptosis. The problem with PI/7AAD is that they MUST remain in the buffer for analysis. Therefore, cell fixation is not an option. Coupled with the reversible binding property of Annexin V, the assay requires careful planning and is more time-sensitive than other assays that allow fixation to preserve the cellular status.

 

There are many ways to detect cellular apoptosis. For example, you can perform western blot analysis to detect the activation of pro-apoptotic caspase enzymes. In addition, you can also perform “tunnel” assay to look at double strand DNA fragmentation, which is an indicator of late-stage apoptosis. However, annexin V staining is by far the most straight forward and requires less time. First of all, annexin V staining looks at the early stage of apoptosis and simple staining of PS on the cell surface. It requires less incubation time as compared to “tunnel assay”, which require incorporation of enzymes to label the broken end of double stranded DNA. So, next time if you are thinking about studying programmed cell death, give annexin V staining protocol a try!

References:

  1. Vermes I, Haanen C,Reutelingsperger C. (2000) Flow Cytometry of Apoptotic Cell Death. J ImmunolMethods 243(1–2): 167–90.
  2. BD Bisosciences. Support Protocols: Annexin V Staining Protocol

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