We are measuring Annexin V by flow cytometry using human platelets. We are doing the activation in PRP prepared from blood containing heparin as the anticoagulant. We have chosen to activate the platelets with convulxin as a positive control for our assay. The problem is that we only have about 7% of positive cells at the highest concentration of convulxin used. That concentration is supramaximal when we measure CD62P and PAC1. Does someone has experience with annexin V measurement by flow cytometry? Is there a better agonist that we could try instead of convulsion? What is the type of values that we should expect for maximum activation?
Thanks
I did the expt with washed platelets. I try to adjust the mean fluorescence in the FL-1 histogram at 1. In a well-prepared platelet population the percentage of platelet showing Annexin V binding should not be more than 3%. If >5% cells in the population are showing annexin V binding then the platelets are pre-activated.
The highest value of Annexin V binding in my expt was 78% cells in the population with 1uM ionomycin and 0.5U/ml thrombin alpha. I have seen Rand et al. reporting 100% annexin binding in isolated platelets.
Thanks a lot for your answer. I will try ionomycin as a positive control. The baseline value for annexin V in our experiment is around 1% so I think the platelets are in good shape.
In our experiments, both ADP and TRAP have activated platelets and increased the Annexin V values.
Could you clarify whether you are attempting to measure platelet annexin V, or are you using fluorescently labelled annexin V as a probe to detect platelet surface phosphatidylserine exposure?
- Badges
- Science method