I am doing an apoptosis analysis by flow cytometry. The cells were stained by AnnexinV and DAPI. The DAPI negative cells show an decreased FSC and increased SSC, suggesting an apoptotic phenotype. But the cells do not show increased Annexing V compared to the control cells. Anybody encountered that before? What is the possible explanation for that? This was not an reagent issue because the DAPI positive population were AnnexinV high, so the AnnexinV reagent was working.
Hi,
Itseems like Annexin V is working but when you say you have not found any difference in Annexin V compared to the control cells that indicates either your treatment (Drug or any additional factor) to induce apoptosis has not worked or has no effect on Apoptosis.
There can be necrosis also occuring in the cells not just apoptosis. To rule out if necrosis is taking place, you can repeat the same experiment but with Propidium Iodide staining and Annexin V.
Annexin +/ PI- = early apoptotic, Annexin +/ PI+ = Late apoptotic
Annexin-/PI+ = Necrosis, Ann -/PI - = Alive cells
All the best.
Shalini Suresh
You must take account early apoptotic and late apoptotic cells. Annexin V stains phosphatidylserines. PS are normally inside of the membrane but due to membran scrambling during apoptosis they thrown out to outer surface of cell membrane. That shows you early apoptosis, because membrane integrity wasn't unbroken yet. At the late stages of apoptotis scrambling activity will be higher and membrane integrity will be broken. At this stage DAPI works and can stain the nucleus. (DAPI, PI, 7-AAD are same..)
Could it be a timing issue? If the incubation time exceeds the amount of time for your treatment to induce apoptosis, then the plasma and nuclear membranes may be too degraded for DAPI or AnnexinV to remain bound. Your cells from the same time point which are DAPI+/AnxV+ may be at lower doses or treated with less toxic conditions.
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