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effect of calcium and magnesium during PBMC isolation?


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#1 hoshi

hoshi

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Posted 20 August 2009 - 08:26 AM

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?
Thanks :lol:

#2 Kristina @Invitrogen Dynal

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Posted 04 September 2009 - 05:44 AM

Hi Hoshi

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.

Kristina




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