Hello everyone,
Should we be preparing 1X concentration of Annexin V binding buffer just before the experiment or Is it okay if we use previously made cold 1X binding buffer?
I've two contradicting results and I wonder if this is the reason. The Annexin staining is much weaker when I used previously made cold binding buffer.
I always prepared Annexin V binding buffer the day before I was going to prep my cells for staining and same day FACS analysis. I usually made a small amount of sterile 10x binding buffer and then diluted it to a 1x concentration. Any remaining 1x binding buffer not used was discarded. I would encourage you to make fresh binding buffer every time, to ensure the potency of the buffer and enhance the accuracy of your results (especially since you see variations in result runs).
While its better to prepare fresh binding buffer every time for your experiment, couple of times I have used pre-prepared binding buffer too and my experiment worked just fine.
Hi Siva, I have used old Annexin V buffer at RT without an issue as annexin V binds at RT not when cold so this is the reason why your old cold buffer did not work as well. After all annexin V buffer is an isotonic salt solution with high levels of calcium so should not go off. I have kept old buffer out on the bench at RT foe weeks without a binding issue.
Regards
Gary
Hi! I used that assay pretty often and I have never had problems using previously prepared annexin buffer. I let it sit until it reaches room temperature to use it.
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