Our lab is working on PBMC cell for compound screening. We are surprised by finding that the level of CD69 in PBMC is very high prior to IgM stimulation(50-60% or so), and after IgM stimulation, the level of CD69 is further increased, we could calculate IC50 of compounds by our protocol, but still very much concerned on the basal level of CD69 in PBMC. Is this because of the serum we used that was not heat inactivated(56°C, 30mins), or any other reasons?
Dear Dr. Renshan Zhang,
Mitogenic stimuli such as concanavalin A, pokeweed mitogen, phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) is used to check for the proliferative responses of PBMCs.
CD69 is also one of the earliest activation markers to appear on the surface of activated lymphocytes. Resting peripheral blood lymphocytes express almost undetectable basal levels of CD69 but addition of mitogenic stimuli results in rapid (2–4 hours) induction of CD69 expression.
Have you added mitogen to the culture?
You may want to refer to the article attached below.
https://www.sciencedirect.com/science/article/abs/pii/S1465324907700701#:~:text=CD69%20is%20also%20one%20of,expression%20%5B11%2C%2012%5D.
Regards,
Malcolm Nobre
Malcolm Nobre Many thanks! We did not add mitogen into the culture, and the PBMC was newly thawed, in which the CD69+ is so high. More recently, we find after 24hrs incubation, the amount of CD69+ was significant reduced. Perhaps, the stress incurred during thawing of PBMC might stimulate the CD69 expression.
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