Hi Dr. Adon,

The two technologies detect apoptosis from different angles and stages, which may lead to different results. And the focus of the question you raised should be the difference between flow analysis and staining analysis. Let's assume compound A and analyze its corresponding results in the two methods.

1. In flow cytometry analysis, generally uses the PI (if it is wrong, you can correct me) to determine the DNA content.

It is generally recognized that the characteristic of apoptotic cells is to maintain membrane integrity. So we will choose DNA breakage as an indicator to indicate apoptosis. This leads to the fact that the flow cytometry results reflect the accumulated apoptotic cells, and cells with membrane rupture in the late stage of apoptosis (DNA is also broken) may also be taken into account.

2. In AO/EtBr method: AO (acridine orange) labels live cells (green fluorescence, intact membrane); EtBr (ethidium bromide) only enters cells with damaged membranes (red fluorescence, late apoptotic/necrotic cells).

Live cells: AO+ (green), EtBr-;

Early apoptosis: AO+ (nuclear fragmentation, green), EtBr-;

Late apoptosis/necrosis: AO+ + EtBr+ (red or orange-red)

We can see that AO/EtBr can clearly distinguish the early and late stages of apoptosis, because the two stages correspond to different staining results. And the apoptotic cells reflected by AO/EtBr can be regarded as a process quantity in the flow cytometry results, which is not equal to or less than the final quantity of the flow cytometry results.

So, if compound A induces rapid apoptosis, cells quickly enter the late stage (membrane damage) or secondary necrosis, AO/EtBr may miss these cells (because EtBr staining also marks necrotic cells) (AO/EtBr counts are low), while flow cytometry can still detect the sub-G1 peak of DNA degradation (flow cytometry technology is considerable).

If compound B induces slow apoptosis, more cells remain in the early stage (intact membrane, AO + green), then the AO/EtBr count is higher.

If the characteristics of compound A cause the AO/EtBr count to be low enough, it may happen that compound A originally performed better than compound B in inducing apoptosis in flow cytometry, but after analysis with AO/EtBr, the AO/EtBr count may not perform as well as compound B.

The above are my personal analysis, mainly based on theory. If you have other opinions, please continue to discuss with me.

Hmmm...So, seeing different results between the flow cytometry sub-G1 peak and the AO/EtBr apoptosis assay isn't unusual. Think of it like tracking a demolition. The sub-G1 tells you how much debris (fragmented DNA) is around, which is a sign of the building breaking down. But AO/EtBr is like looking at the building's structure and whether its walls are still intact while the inside is collapsing.

Different drugs can make cells die in different ways and at different speeds. One drug might cause a lot of DNA damage quickly, leading to a big pile of debris (high sub-G1), but the cell's outer walls might fall apart fast too, so you don't see many "early damage" signs with AO/EtBr. Another drug might cause a slower, more structural collapse that you catch earlier with AO/EtBr before the DNA really starts to shatter. To really understand what's happening, it's best to check at different times and use a few different ways to look for cell death, like having multiple ways to inspect that collapsing building

Attapong Sinkitjasub . Thank you for generously sharing your knowledge and for explaining things so clearly. I truly appreciate your guidance. If it’s not too much trouble, could you kindly let me know about other methods available to assess or confirm cell death?

Lucian Miles. Thank you for sharing your knowledge with me—it has really helped clarify my understanding and will greatly support me in interpreting my results.

I have a question: when analyzing apoptosis using AO/EtBr staining, is it better to report the different stages of apoptosis (early vs. late/necrosis), or should I just count the total number of apoptotic cells, regardless of the stage? I’ve noticed that many studies report the stages separately, but my co-guide advised me to show only the total apoptotic count, as he feels AO/EtBr isn’t reliable enough to distinguish early and late apoptosis. What do you suggest?

"That's a great next step in thinking about how cells die! Besides looking at fragmented DNA (sub-G1) and early cell changes (AO/EtBr), we have other ways to check. Think of it like having different tools to see if a lightbulb is broken: 1) Annexin V/PI: Checks if the cell's outer layer is starting to get messy (early sign of death) and if it's completely broken (later stage)., 2) TUNEL: Directly looks for chopped-up DNA inside the cell, a clear sign of cell death.,

3) Caspase Assays: Detect special helpers inside the cell that get activated when it's told to self-destruct., 4) Mitochondrial Assays: See if the cell's energy source is failing, which often happens early in cell death., and 5)Viability Assays: Generally tell us if the cell is alive and healthy or not. (For more methods, you'd better consult your senior lab researchers or advisor for more suitable for your lab setup and your own research environment.)

Each of these methods looks at a different part of the cell death process. If you want to dig deeper into how each one works and what kind of information they give us, I encourage you to look them up! Understanding these different techniques will give you a much clearer picture of what's happening to the cells." Good luck :)

Attapong Sinkitjasub .. Your suggestion gave me a clear understanding of how I should proceed further. Thank you.

Yes, Tenzin Adon. Your mentor's suggestion is very scientific.

Because AO/EtBr staining has limitations in distinguishing apoptosis stages.

Although in principle, we can distinguish different stages based on the following characteristics:

Early apoptosis: AO penetrates the intact membrane, and the nucleus is green, condensed or crescent-shaped (PMC4332266).

Late apoptosis: EB infiltrates, and the nucleus is orange-red fragments (PMC4332266).

Necrosis: EB is uniformly orange-red, and the nuclear morphology is intact.

However, there are some special cases, such as EB may infiltrate mechanically damaged cells (such as over-digestion), which will be misjudged as late apoptosis.

It is also possible to make low-level mistakes, such as confusing nuclear fragments with intact nuclei, resulting in incorrect counting.

I would also like to remind you to pay attention to your apoptosis inducer and at which stage it causes cell death. If there is an early apoptosis inducer, it may not be suitable for general counting of the total number of apoptotic cells.