Does anyone have experience of using plasma that has been anticoagulated
by citrate (as is routine for blood donation in the UK) for the purpose
of addition to MLR? I presumed it would be best to reverse the calcium
deficit and allow clotting to occur. To do this required addition of
calcium chloride in quite large amounts (500 ul of 0.26 M solution), and
inevitably there was an over shoot. The plasma clotted okay and left
clean serum, but the measured calcium concentration was 14 mM. When
tested in an MLR the serum appeared to allow normal proliferation at
concentrations up to 10 percent, when presumably the rise in medium
calcium would be relatively insignificant. However, I am interested in
using higher concentrations of the serum and at concentrations of 20
percent or higher the calcium concentration will be way outside the
normal range. So far it looks as though this promotes even higher
activation of T-cells. Interestingly I cannot find any publication
listing the effect of high extra-cellular calcium concentration on T
cell activation on Medline (I guess relevant publications may pre-date
Medline), but from the knowledge that calcium influx is crucial to
T-cell activation I presume the effect would be enhancing. I tried
adding EGTA, sufficient to lower the measured calcium to 4 mmol, but
this seemed to completely stop T-cell proliferation, and this effect of
EGTA is well described in the literature, and presumably that means the
actual free calcium was lower than that measured by our standard
biochemistry lab assay. Anyone have any comments or thoughts on this
problem?
