I want to check the effect of IL2 on the phenotype of a population of mouse CD8 T-cells I'm studying. So I want to stimulate my cells with IL2, anti-CD3 or anti-CD3+IL2. Recently I've ordered 100 mcg human recombinant IL2 from Peprotech.
In the manual it's suggested to dissolve powder in 100 mM sterile acetic acid as 1 mg/ml. I equilibrated temperature of the vial, centrifuged, dissolved in 0.1 M acetic acid, waited for 3-5 minutes to give better solubilization. Then I dissolved my protein solution additionally to 50 mcg/ml in sterile PBS/0.1% BSA, aliquoted and froze at -80. 1 vial was diluted more in order to get 20000 U/ml as the instruction claimed IL2 activity to be 10 millions U/mg. So as I got 100 mcg IL2 it means I got 1 million units IL2.
But this IL2 added to get 50 U/ml doesn't support CD8 T-cell survival neither alone nor during polyclonal T-cell stimulation and even decrease their survival what is strange.
Why it is so? Any ideas? Any suggestions?
Many thanks!
Cell stimulation protocol is like that:
Sorted CD8 T-cells are either cultured at IL2 50 U/ml or at plate-bound anti-CD3+soluble anti-CD28 or at plate-bound anti-CD3+soluble anti-CD28+IL2 50 U/ml.
As I tried before IL2 induced my CD8 T-cells to proliferate even at 25 U/ml.
Now it is not only supportive alone but it even reduces anti-CD3 induced proliferation.
Any idea where the mistake can be?