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

“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.

Join Expert Cytometry's Mastery Class
Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

Similar Articles

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

By: Tim Bushnell, PhD

Reproducibility has been an ongoing, and important, concept in the sciences for years.  In the area of biomedical research, the alarm was sounded by several papers published in the early 2010’s.  Authors like Begley and Ellis, Prinz and coworkers, and Vasilevsky and colleagues, among others reported an alarming trend in the reproducibility of pre-clinical data.  These reports indicated between 50% to almost 90% of published pre-clinical data were not reproducible.  This was further highlighted in the article by Freedman and coworkers, who tried to identify and quantify the different sources of error that could be causing this crisis.  Figure 1,…

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

By: Tim Bushnell, PhD

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

Flow Cytometry Year in Review: Key Changes To Know

Flow Cytometry Year in Review: Key Changes To Know

By: Meerambika Mishra

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

What Star Trek Taught Me About Flow Cytometry

What Star Trek Taught Me About Flow Cytometry

By: Tim Bushnell, PhD

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

5 Flow Cytometry Strategies That Sun Tzu Taught Me

5 Flow Cytometry Strategies That Sun Tzu Taught Me

By: Tim Bushnell, PhD

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

By: Meerambika Mishra

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest.  Cells Flow cytometry measures biological processes at the whole cell level. To do…

Which Fluorophores To Use For Your Microscopy Experiment

Which Fluorophores To Use For Your Microscopy Experiment

By: Heather Brown-Harding, PhD

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

4 No Cost Ways To Improve Your Microscopy Image Quality

4 No Cost Ways To Improve Your Microscopy Image Quality

By: Heather Brown-Harding, PhD

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

By: Heather Brown-Harding, PhD

TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment.  TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch.  What does it mean in practice?  A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.