I would like to evaluate the phosphorylation of Akt in human peripheral blood lymphocytes using western blotting or flow cytometry.
I used PMA/ionomycin or CD3/28 Dynabeads (ThermoFisher) as a stimulant, but could not obtain a positive control.
If you know some stimulants that are useful for phosphorylating Akt in human lymphocytes, I am very happy to be told about them.
PMA/Ionomycin and LPS from E.coli could be used as stimulants. what was the time period you used. I saw activation at 15 mins and 30 mins post stimulation of PBMCs.
You might try lithium chloride (Licl), an activator of Wnt signaling pathway.
I usually used LiCl at a concentration of 2-5mM for 30-60 minutes for stimulation.
Best regards,
Christian
Crosslinking BCR of B cells with anti-IgM might do the trick. Anti-CD3/CD28 antibodies should work too - no need for fancy beads to get involved. Good luck!
Thank you for answering my question. I will try following your advices.
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs