While performing Annexin V and 7 AAD for identifying early and late apoptosis, what would be the ideal control? Is it recommended to add Annexin V and 7 AAD in the untreated samples and negate the values from the test? As I see many cells (around 60%) are undergoing early apoptosis (Annexin +) without any treatment at all. If I use this as the control to negate the values, then my actual treatment causes only 10-15% apoptosis. While when I use unstained as my control, apoptosis in my treated sample shows 70%+. I did not come across any article where they use Annexin V and 7 AAD in the untreated samples as well. Is it possible that these are adherent cells and are likely to have damaged cell surface due to trypsinization? I used HS5 cell line.
Dear Sowmya,
Annexin V bind to phosphatidylserine that, in apoptotic cells, is translocated from the internal to the external cell membrane layer. 7-AAD intercalates ds DNA in necrotic cells. So, if you use untreated cells as a negative control, you should consider to use any obtained signal as noise and to use it to remove the same value to the treated samples during data analysis. In this way, you will get a clean signal.
I hope to have been useful, good luck with your job.
Davide
Dear Sowmya,
You should first set a gate with unstained cells to avoid autofluorescence; cells should be processed similarly as that of your experimental samples excepting that they are not getting the stains. Next use the untreated controls but stained as your experimental control to compare between the samples.
As you have mentioned that around 60% of untreated (but stained) cells are undergoing early apoptosis, the reasons could be anything happening during the processing (staining) steps. Be very careful in handling the cells during the staining procedures because they will be very vulnerable and mishandling might induce apoptosis even if they are untreated.
Hope this helps and good luck with your experiments.
It is surprising that 60% of untreated cells are Annexin positive. You can try a more gentler way of cell detachment. Accutase will be an option. It has been used with great success in dissociating iPSC colonies as well as primary cultures. Now you know where to ask:). If your pipeting is not harsh then you should check for mycoplasma as well. If it is a primary culture then you may want to use cells at earlier passages for the experiment and check if it makes a difference.
if you are using cells in early passages uch aspassage 1 or 2 you will get this results, this is due to fragile cells in these passages
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