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How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

Written by Tim Bushnell, PhD

“If one approaches a problem with order and method there should be no difficulty in solving it — none whatever.”

— Hercule Poirot, Death in the Clouds

Murder is a common theme in the mystery suspense genre. The detectives who solve these murders use a combination of observation and deduction to identify the guilty party. This metaphor suits measuring cell death.

In biology, there are four major pathways for cell death.

The study of the different ways cells die has become known as Cell Necrobiology, as coined by Darzynkiewicz and coworkers in their 1997 review article.

Flow cytometry is ideally suited as a tool to study Cell Necrobiology and, with its plethora of reagents, it is even possible to follow the different steps in these processes.

The four major ways a cell can die are:

Cell death is so important, that it has been the center of several Nobel prizes including one awarded in 2002 to Sydney Brenner, Robert Horvitz, and John Sulston who discovered the genes involved in apoptosis, and again in 2016 when Yoshinori Ohsumi was recognized for his work on the mechanisms of autophagy.

Of these mechanisms, apoptosis is probably the most readily studied using flow cytometry.

There are many assays that can be performed to measure apoptosis in cells. These can be grouped by the state of the cell as it dies.

1. Measuring apoptosis.

One of the first stages of apoptosis are changes seen in the mitochondria, where the membrane potential collapses, which leads to the release of several factors that can inhibit anti-apoptotic proteins, as well as the release of cytochrome c, which binds to another protein that ultimately causes the activation of caspase-9, and in turn caspase-3.

There are a host of dyes that can measure this depolarization, including CMXRos, JC-1, and TMRE.

One of the most common is JC-1, which is a cell permeant dye that emits a red fluorescence (~590 nm) in healthy, active mitochondria because of the formation of aggregates. As the membrane potential collapses, the aggregates fall apart and the fluorescence shifts to a green color (~529 nm).

This is observed by flow cytometry as a change in the ratio, as shown in this data from Derek Davies, head of the flow cytometry facility at the Francis Crick Institute in London.

Apoptosis JC-1 monomers measured with flow cytometry

Here, untreated cells show a dominant red fluorescence, but after drug treatment, there is a dramatic shift to green fluorescence.

The next steps in apoptosis include the activation of the caspases, and changes in membrane symmetry and permeability.

Phosphatidylserine (PS) is found on the inner leaf of the plasma membrane. As apoptosis progresses, PS flips to the outer membrane, which is a signal for the cells to be phagocytosed.

The protein Annexin V is a calcium-dependant protein that preferentially binds to PS. When you add a cell-impermeant dye, such as 7AAD or PI, you get a very robust assay for looking at apoptotic and necrotic cells. Typical data are shown below:

Annexin V is a calcium-dependant protein that binds to PS

Annexin V is calcium-dependant and not very stable. In general, it is best to read Annexin-stained cells within an hour or so of staining.

If you’re planning to perform a lot of Annexin assays, this buffer works very well:

10x Annexin Buffer

0.1 M HEPES

1.4 M NaCl

25 mM CaCl2

You can stain cells with surface markers, which is best done before staining for Annexin V.

2. Measuring necrosis.

One of the hallmarks of necrosis is the loss of membrane integrity, which leads to the easy use of a host of cell-impermeant dyes from PI to 7AAD and others.

In the Annexin assay above, cells that are Annexin negative and DNA dye positive are often considered to have died by necrosis. Unfortunately, these cells can also show up in the Annexin positive, DNA dye positive fraction, making it an imperfect measure of necrosis.

It turns out that high-mobility group B1 protein (HMGB1) may be able to differentiate necrotic cells.

This nuclear protein stays contained within the nucleus during apoptosis, but is released when cells undergo necrosis (Raucci et al., 2007). Currently, this is typically done by analyzing the supernatant for HMGB1, or by microscopy.

This would be an excellent assay to implement on a tool like the ImageStream.

3. Measuring autophagy.

Historically, autophagy has been defined by the measure of the ‘autophagosome’ by either electron or light microscopy.

The marker LC3 could also be used in microscopy techniques, especially since LC3 was best measured when it was tagged with a fluorescent protein like GFP.

Fortunately, a paper was recently published by Chikte and co-workers (2014), in which the authors report the use of the dye Lysotracker Green DND-26 as a way to measure autophagy in cells.

They compared this dye to LC3 and demonstrated that Lysotracker gave similar results, and importantly, was easier to use than LC3.

Using flow cytometry and a host of different reagents, it is possible to tease out how your cells may have died. Like the most famous consulting detective once said, “When you eliminate everything else, that which remains, however improbable, must be true” (Sherlock Holmes, the Sign of Four). Using these tools, you can readily eliminate the various suspects and come to your conclusion as to how your treatment may have killed your cells of interest.

If you’re interested in further reading, this review article by Wlodkowic and coworkers (2011) Methods Cell Biol 103:55-98 is an excellent reference.

To learn more about how to use Flow Cytometry to measure Apoptosis, Necrosis, and Autophagy, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Tim Bushnell, PhD

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