I'm aware it's not recommended to thaw and refreeze bfa but I prepared larger volumes of aliquots. I would really like to be economical and use it at least once after thawing, but I was hoping if anyone who has undergone freeze-thaw once and used it in intracellular cytokine staining can share their experience. Does reusing an aliquot makes any difference in terms of interferon gamma responding T cell proportions detection by ICS?
In my experience, it is perfectly fine to freeze-thaw brefeldin A aliquots for a limited number of times. In our lab, we limit the number of freeze-thaw cycles to 3, after which any remaining volumes are discarded. By estimating the volumes required per experiment, it is possible to plan optimal aliquot volumes. Using this strategy, we have never seen any variation in ICS IFN-gamma responses.
Another important factor is the storage temperature. While product sheets of brefeldin A, and several other products, indicate -20°C to be suitable for storage, it is actually much better to store these reagents at -80°C. Compared to -80°C freezers, -20°C freezers freeze the reagents more slowly and also show greater temperature fluctuations during storage, together resulting in faster reagent degradation.
Great! I'm happy to learn that it can be used more than once. Thanks a lot Wim and Robin!
We actually dissolve it in Methanol aliquot to 200 - 250 µl and store at -26°C . . . . no freeze/thaw cycles. Dissolved and Stored this way it's good for at least a year (I don't think we have ever gone longer yet without having to make a new batch).
We dilute Brefeldin in DMSO which seems to turn the solute more stable. As freezing temperature of DMSO is about 18ºC, freezing in -20ºC freezers usually froze the aliquot of Brefeldin almost instantaneously decreasing the degradation.
In this condition we usually freeze-thaw until 5 times without evident degradation or variation of the ICS assays.
I appreciate the feedback Hans-Peter and Dewton.
My stocks are frozen down in DMSO and stored at -20. It sure is a relief to know that it can be freeze-thaw more than once.
As already described in the previous posts no matter in what it is dissolved it’s going to be stable for at least 3-5 thawing cycles. Personally I’m dissolving it in DMSO and store it at -20 for more than a year but a problem with thawing might occur with Ionomycin. It is less stable an you should not thaw it more than 3 times!
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