Quantification of tumour cell—endothelial cell attachment by 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)

Tumour cell attachment to the endothelial cell lining of the circulatory system is of utmost importance in the process of cancer spread. We describe here a method of quantifying tumour cell attachment to an endothelial cell layer in vitro, using the fluorescent carbocyanine dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI). We show that by incubation of human tumour cells with this fluorochrome, a high degree of fluorescent label can be incorporated into the cells without cytotoxic effects. These labelled tumour cells can then be used in subsequent attachment assays involving confluent human endothelial cell layers and subsequently quantified by using a fluorescent plate reader. Monitoring of this assay by fluorescent microscopy showed no transfer of the dye between tumour and attached endothelial cells. The labelled cells remained fluorescent for more than 3 days with no observable cytotoxicity. We suggest that DiI is of use in an assay system such as this to determine the effects of various factors on tumour cell-endothelial cell attachment.