I tried Annexi V staining coupled with FITC flouroscent stain and analysed it by flow cytometry. I tried these two buffer compositions:
Buffer1: 10 mM HEPES/NaOH, (pH 7.4), 150 mM NaCl, 5 mM KCl, 5 mM MgCl2, and 1.8 mM CaCl2
Buffer2: 10 mM HEPES (pH7,4), 140 mM NaCl, 4 mM KCl, and 0.75 mM MgCl2
However, none of them worked. The problem is Annexin V stained positive cells are found only in the region where cell debris are found. To explain the problem I am attaching this image which gives a comparison of Annexin V positive cells when considered different population of the same run.
The cells did undergo apoptosis as the apoptotic inhibitor protein was knocked down. All the cells died at 72 h hence I harvested them at 48 h and the results are shown here.
Could the problem be the duration of your culture? I have used Annexin:FITC combined with propidium iodide (for necrotic cells) in the past and got the best results after about 16 hours (for T cell mediated killing of target cells). Once the cells start dying (become PI+) it becomes increasingly difficult to analyse them. We always focused on the Annexin+PI- ("early apoptotic") population. Maybe a time course with these two dyes will lead you to a better time point for analysis.
The Annexin V bining buffer I used was: 10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2.
Another point I always thought is crucial is to account for all the fully disintegrated cells by doing total cell counts at the beginning of the experiement and at the time of the Annexin staining.
Good luck,
Axel
Thank you vey much Axel. I will try analysing the cells at shorter time points.
Most important thing is to include Ca2+ in the binding buffer (any isotonic buffer) without which Annexin V will not bind PS. Since secondary necrosis always follows apoptosis and all cells may not undergo apoptosis at the same time, it is better to establish an optimal time point initially by performing a time course analysis.
Best,
PS
Yes, the compensation must be adjusted. It can easily be done in FlowJo.
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