I am working on an in vivo experiment in rats where I implanted (subcutaneously) genetically modified C2C12 murine myoblasts that deliver Epo enveloped in a delivery system we are working on in our lab.
Some of the experimental groups were transiently immunosupressed and others weren’t, in order to evaluate the protection our delivery systems offer by themselves.
In addition to histological evaluation of the implants (at the end of the study) and hematocrit values during the study period, I would like to find out which are the exact reasons for the system to fail.
My problem is that I know what I want to find out but don’t know which molecules/cells I should look for and which techniques I should employ (as this is the first time I am doing this and after having a look at other publications, I can’t find a common idea).
As expected, the non-immunosupressed group’s hematocrit values have already started to drop while the immunosupressed group still shows high values.
I would like to evaluate on rat plasma/serum the following:
1. Did the rat generate cytokines which are attacking the murine C2C12 and this leads to a lack of Epo production? (which cytokines would be more suitable to measure, consdiering we have performed a SC implantation?)
2. Did the rat generate anti-murine-Epo antibodies that are blocking the Epo being delivered by the C2C12 and thus hematocrit levels start to drop/do not rise any more?
What is most appropriate to measure cytokines and Ab-s? Serum or Plasma? or any of them?
Thank you very much to everyone who may give me any ideas. I need to step forward and it’s getting difficult!!!
As far as I know, you should be able to detect both ab and cytokines from serum as well as from plasma. However, at least cytokines can be difficult if not impossible to detect that way due to their small concentrations, and the results can be quite ambiquous. Anyways, if you manage to detect cytokines, I’d probably go for Th1 vs. Th2 cytokines for example, to evaluate the function of T-cells, which are responsible for the graft rejection. Antibodies should be more straightforward to measure, probably with some ELISA system.