Hi
I'm trying to reconstitute IL-2 from Miltenyi Biotec. The protocol from Miltenyi recommends:
" It is recommended to reconstitute lyophilized Human IL-2, premium grade with deionized sterile-filtered water to a final concentration of 0.1–1.0 mg/mL in a minimal volume of 100 µL. Further dilutions should be prepared with 0.1% bovine serum albumin (BSA) or human serum albumin (HSA) in phosphate-buffered saline."
I assume the lyophilized product has some buffer, salts and stabilizers but has anyone actually gone along with this protocol? Also in order to dilute it in RPMI do I still need to dilute it down in PBS first? Doesnt look very logical !
thanks !
The recommendations are to minimize product loss and degradation. They recommend a minimal volume of 100 uL for reconstitution so you can minimize the cytokine sticking to the vial after reconstitution and when you aliquot. You tend to lose more when working with small volumes that are highly concentrated. The lyophilized cytokines do come with stabilizers but the more you dilute, the stabilizers also get diluted out. So if you plan to make working stocks and freeze them, the manufacturers recommend that you dilute stocks in PBS containing serum, to minimize degradation and product loss over time. I have done working stocks in sterile water and frozen those. Although I never had issues, I cannot guarantee this.
You could just choose to freeze reconstituted stock solutions and make working stocks when you need to, in RPMI. The problem is that usually you use cytokines at the ng/mL range and you need to dilute the stock quite a lot to get to your working concentration. In doing so, you can’t refreeze the diluted cytokines (in RPMI) and you would end up having to throw away the unused cytokine*. So it is really not economical. The best thing to do would be to make single use aliquots of a reconstituted stock (0.1 -1.0 mg/mL) and working stock (1-10 ug/mL). Whether you choose to do this in PBS. BSA or sterile water is up to you.
*I also have been in labs where we had M-CSF resuspended in RPMI and frozen at -80. But again, no idea how long it stays active for and how many freeze-thaw cycles you can subject it to.
fantastic thanks a lot for your input. I think I'll go with your suggestion. I'll use deionized water for the primary stock and make loads of aliquotes. Then try to make working stocks with PBS etc and if needed refreeze those.
cheers
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