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7 Tips For Measuring And Reporting Apoptosis By Flow Cytometry

Written by Tim Bushnell, PhD

Cell death is a fact of biological life.  How, when, where and most importantly, why cells die, can have huge biological consequences on the path an organism may take.

Apoptosis, or programed cell death, can result in a selective advantage for an organism. Fingers, for example, are the result of apoptosis of cells during development.

Next to immunophenotyping, measuring apoptosis using flow cytometry is one of the most common assays. It may be because of the many different ways to measure the process, many of which can be easily performed in a high-throughput manner, or combined with other assays to determine if specific cellular subsets are sensitive to a given drug or treatment.

This subject was so critical to understanding cellular functions that the Nobel Prize committee recognized the study of apoptosis as seminal and in 2002, the prize in Medicine was awarded to Sydney Brenner, Robert Horvitz and John Sulston for their work on the subject.

7 Apoptosis Tips

Annexin V is a calcium dependent protein that binds preferentially to phosphaditylserines. These phospholipids typically are inward facing, but are flipped to the extracellular side of the membrane as one of the early signals of apoptosis. Coupled with a membrane impermeant dye (like 7AAD or PI), early and late apoptosis can be characterized.

Here are 7 tips for performing your next apoptosis assay using Annexin V and a membrane impermeant dye:

1.  Make sure you use the correct buffer. 

Annexin V is dependent on calcium for binding. When performing this assay, make sure to use a calcium containing buffer during the labeling process.

2.  Annexin V binding is not stable. 

Unlike antibody binding, the Annexin binding is not that stable. It also doesn’t fix well. Thus, it is critical to read these samples shortly after labeling – typically between 1 to 3 hours from the point of labeling.

3. Pick the right assay.

Inducing apoptosis can be done with a host reagents. Make sure the reagent being used induces apoptosis and not necrosis. Sub-G1 fragmentation is an oft mis-used method for detecting apoptosis. In this assay, low-fragment DNA is measured by standard DNA binding dyes, appearing to the left of the G1 peak on a DNA histogram. However, if the assay is not performed correctly, estimates will be off.

4. Watch your fixation.

Formaldehyde fixatives tend to cause the small DNA fragments to be retained, and thus underestimating the apoptosis.

5.  Consider TUNEL.

TdT-mediated dUTP nick end labeling. Using terminal deoxynucleotidyltransferase (TdT), the ends of DNA breaks are labeled with dUTP that can be later detected using an anti-BrdU antibody. In this case, you do need to use formaldehyde fixation. You also don’t need to perform DNA denaturation, as the smaller fragments are readily accessible to the antibody. Loss of mitochondrial membrane potential is another early hallmark of early apoptosis. In cells undergoing apoptosis, the mitochondria will release cytochrome C and the apoptosis inducing factor – both of which are necessary of caspase activation (another critical step in apoptosis).

6. Choose your dye carefully.

Three common dyes are Rhodamine 123, 3,3’-dihexyloxacarbocyanine iodide, and JC-1. R123 should be coupled with a dye like PI so that live (cells staining with R123), early apoptotic (cells lost the ability to accumulate R123) and late apoptotic (PI positive cells). JC-1, on the other hand goes from a green fluorescence to an orange fluorescence in cells undergoing apoptosis.

7. Don’t over-interpret the data.

Some papers suggest that the collapse of the mitochondria membrane potential may not be a critical step in apoptosis. The list of possible assays goes on. Detection of activated caspases, either directly with antibodies, or by activity, can be a very informative assay as well. In the end, each assay offers strengths and weaknesses. Understanding what the output is and how the assay is to be combined with other readouts. Likewise, knowing a bit about the process of apoptosis in the cells of interest is critical.

To learn more about performing apoptosis assays by flow cytometry, including how to publish your apoptosis data, join our special webinar Thursday, November 6th: How Measure, Analyze, And Publish Apoptosis By Flow Cytometry

Tim Bushnell, PhD

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