I am trying to design a experiment in rat to look at the effect of tumor inplants on the maturation and Antigen presenting capacity of Dendritic cells isolated from tumor bearing rat, and whether feeding the rat with certain plant extract could reverse the tumor effect on these cells (if any).
My questions are as follows:
1. I am aiming to isolate DC from PBMC of rat for these assays after sacrifice. Will there be enough DC to be isolated from PBMC for these assays, or do I need to generate them from the rat bone marrow? The only concern I have for the latter approach is that any tumor induced effect could be reversed during culture in the absence of the tumor.
2. I want to activate DC with known antigen (viral or tumor associated) to induce maturation in vitro and look at their CD80 and CD86 expression with flow cytometry. Will DC be activated to mature by co-culturing DC with the purified antigen (or peptide)? or do I need to add any stimulant such as LPS.
3. If I want to measure the IL-12 secretion by DC, do I simply co-culture DC in the presence of the antigen? or does in vitro stimulation of IL-2 secretion requires presence of T cells (CD40L-CD40 interaction)?
Thanks very much
Edited by halls, 30 July 2009 - 03:40 AM.