cells "settling" in the tubes
ZBIGNIEW DARZYNKIEWICZ (darzynk@nymc.edu)
Sat, 12 Jul 1997 11:42:02 -0500
Tim O'Neil is describing a problem associated with PI staining when
the suspended cells settle rapidly in the tube resulting that few
cells can be measured. Actually, the phenomenon may be very
troblesome when one has limited number of cells in the sample. This
problem is associated not only with gravity sedimentation of the
cells but also with the fact that when cell suspension is transferred
into a new tube the cells rapidly attach to the plastic or glass
surface. Electrostatic forces (electronegativity of the cells
surface) are predominantly responsible for the attachment and the
polystyrene tubes appear to be worse, in this respect, than the
polypropylene ones. The attachment is diminished to the silanized
(silicon coated) tubes. On the other hand the phenomenon is
exacerbated in media of low ionic strength, and since the
commercially available stain solutions keep their composition in
secret, it is hard to evaluate to what extent their ionic composition
contributes to the problem. We have noticed that addition of serum
proteins (e.g. 0.5 % w/v serum albumin) and detergents (e.g. 0.1 %
Triton X-100) diminishes the electrostatic attachment of cells to the
tubing. It should be tested, however, whether albumin has no negative
effect on cells stainability in each protocol. No such effect was
apparent in the case of staining with PI.
Zbigniew Darzynkiewicz
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