Hi everyone!
I'm a PhD student and I've been stuck for long time now with culturing regulatory T cells (Tregs) from the mouse spleen. I've made several attempts but currently I'm using this method below:
-Tregs are isolated via the FACS Aria II (BD) after negative selection of CD4 cells (Stemcell)
- Complete medium used is:
RPMI 1640
10% FBS
50µM Beta-mercaptoethanol
25mM HEPES
0.1 mM Sodium Pyruvate
10U/mL Pen/strep
- As a stimulation method I use the dynabeads (Anti CD3, Anti 28 beads) in 1:2 or 1:3 ratio and 100U/mL or 1000 U/mL IL2 which is added to the cells every two days
- cells are first placed in a 96 round bottom well plate at a concentration of 200,000 Tregs/well
The results I get is that the purity (
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