I am trying to quantify phosphorylation of the p65 subunit of NF-kB in primary murine CD8+ T-cells. I stimulated the cells with plate-bound anti-CD3 (1ug/ml) and anti-CD28 (2ug/ml) Abs for 5min and tried to detect p65 phosphorylation by flow cytometry. I did not detect a signal although I was able to get pErk signal at 5min. Is 5min too early for NF-kB phosphorylation?
Dear Shubhabrata !
Please You look at this article:
Article Anergic CD8+ T Lymphocytes Have Impaired NF- B Activation wi...
Activation of NF-κB is defective in anergic CD8+ T Lymphocytes
Figure 2Anergic T lymphocytes display deficient NF-κB dependent transcription
Purified naive and anergic 2C TCR/NF-κB-Luc transgenic T lymphocytes were stimulated for 48 hours with anti-CD3 and anti-CD28 conjugated beads and luciferase activity was analyzed. **p
Probably the experiment you have to perform is to test a time-course of activation, let say 5, 10, 20, 30, 45, 60, 120 minutes to identify the kinetics of the phenomenon in your system.
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