In our lab we would like to study the chemotaxis of PBMCs towards conditioned medium obtained following treatment of cancer cells with different compounds. My questions are regarding the method of purification of PBMCs from whole blood:
- I have read a number of protocols saying that during the isolation process the buffers used should not contain calcium or magnesium ions, as these can cause the activation of the PBMCs. Is that so? From my knowledge Ca+2 is a second messenger only in TCR signaling. I am not sure reegarding the other cells present, and how magnesium is relevant at all.
- Even if I use PBS (without any calcium or magnesium ions) to isolate the PBMCs from blood, in the end I resuspend the isolated PBMCs in medium which does contain these ions. Is there any rationale to use calcium and magnesium-free buffers for the isolation when the final medium contains these ions anyway?
- When I begin the assay, should the PBMCs be activated or not? In other words, do chemokines also activate the PBMCs or are chemotaxis and activation two totally independent processes? How should activation/no activation affect my results?
Thank you in advance.