Dectection of early apoptosis by AnnexinV-FITC staining - FACS analysis problem SOS!!!!!!!!!!! (Jul/21/2005 )

Hi

I am Hasina * a new member of this forum. I am working with detection of early apoptosis by AnnexinV-FITC staining pattern determined by Fluorescence Activated Cell sorting(FACS}. For this, I use 5% ethanol (and also Fas antibody 500ng/ml}as an apoptotic inducer, trypsinize cells for 3 mins, washed cell dishes with PBS twice to take the cells into falcon tube, centrifuge the cells , resuspend the pellet with 1x Binding buffer, then I add annexinV-FITC and PI, incubate in the dark for 15 mins , then FACS analysis.

But I dont find good amount AnnexinV-FITC positive cells. In necrotic positive sample ( cell dish placed at 80°C for 30min,} I found PI positive cells.But in necrosis I should also found AnnexinV-FITC positive cells, isn"t it?

Do you think the setting of FL1(for FITC}, FL2(for PI} voltages, or compensation is not correct and this is the problem?

or , where is my problem.

I am using Clontech apoalert-apoptosis kit(1xBinding buffer, AnnexinV-FITC, PI} for the experiment.

Please let me know, how can I get rid of this problem, I mean how can I detect early apoptotic cells(AnnexinV-FITC positive} cells?

i would be grateful to you.
regards,
Hasina

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I've also had a bit of difficulty with this kit - problems which were never completely resolved and were put on the back burner for awhile.

The necrotic control should be both PI/Annexin positive (upper right quadrant) while early apoptotic cells should be primarily in the Annexin positive/PI negative lower right quadrant. Do your treated samples test positive for Annexin binding?

If they do not, I would consider troubleshooting the amount of AnnexinV added to the sample. I had similar difficulties obtaining Annexin positive samples, and a fellow from our flow facility suggested adding more to compensate for inefficient binding. Assuming your necrosis control is actually compromising the cell membrane, you should have PS being bound on the inner cytosolic membrane - and cells that are A/PI positive. It seems that the Clontech protocol adds a very small amount of Annexin compared to other kits that are available, and adding more shouldn't negatively impact your experiment.

Let us know how you make out.

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hi,

well, I use the same/similar kit from BD pharmingen and never had any problem
so,

one thing that I concern is your sample might not be apoptotic
use different methods to just make sure your sample is apoptotic

if your sample is apoptotic, maybe your should buy new Annexin.

tk

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few ideas: first of all ethanol rather induces necrosis, not apoptosis. in 10% ethanol 90` - i have 80% necrotic JJK cells. try etoposide or UV treatment, just for the control, to exclude the possibility that you dont actually have an apoptotic population in your sample.
besides PI is toxic for the cells. i usually keep annexin for 15min, but add PI just befor running the sample. it makes a difference.

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