effect of calcium and magnesium during PBMC isolation? - (Aug/20/2009 )
calcium-free, magnesium-free PBS is usually recommended for washing PBMCs during ficoll gradient isolation. Is there a particular reason why it's better to use calcium-free, magnesium-free PBS for this? Is it to reduce cell attachment to the walls of the tubes or prevent activation of monocytes/lymphocytes?
When isolating cells, the blood needs to be treated so as to avoid coagulation during processing. This is necessary whenever processing a sample containing coagulation complement, otherwise micro-clots will form which will impair yield and viability of the isolated cells. Ca2+ and Mg2+ ions play an important role in coagulation activation, and need to be removed, complexed or blocked in order to avoid this problem. There are different ways to accomplish this: Some choose to add EDTA to the sample to bind (complex) the divalent ions, others prefer ACD. If EDTA or ACD is added to the cells during isolation, Ca/Mg-free medium is not necessary.