> Could anyone suggest a method of removing adherant cell lines from culture > vessels which does not remove surface adhesion molecules? At present i am > studying surface cell adhesion molecules expression on human umbilical vein > endothelial cells using flow cytometry. > i would appreciate any suggestions. > thanks > Morgan Sure - we use adherent cells all the time, including HUVEC. Remove the culture medium, rinse with a volume of PBS (that does not contain calcium or magnesium) equal to your culture medium volume. Then incubate the cells with a sterile filtered PBS solution containing 2mM EDTA (this should be added at about one tenth of your culture volume). Incubate for 10 minutes or so - you will probably have to jar the cells loose from the plates. This is not as fast or effective as trypsin for dissociating cells, but it will preserve surface receptors. (The cells dissociate because they require the divalent cations, that are removed by EDTA, for adhesion). You can then dilute the cells in PBS or cell medium depending on what you would like to do with them. If is often helpful to pipette them up and down a few times during this step to disperse clumps of cells and to make sure that they are completely suspended. Sigma also sells something they call "non-enzymatic cell dissociation solution" (which I suspect is exactly the same thing as I described above) if you're inclined to buy that instead. AL
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