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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