Binbin Lee , I have encountered similar questions before.

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 first and analyze its corresponding results in the two methods.

1. In flow cytometry, the dye generally uses 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 the integrity of the membrane. So we will choose the indicator of DNA breakage 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 the dye analysis 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.

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, what you mentioned may happen: A originally performed better than B in inducing apoptosis in flow cytometry, but after AO/EtBr analysis, the AO/EtBr count performance may not be as good as B.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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