Gregory C. Zem

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

Analysis of surface properties of human cancer cells using derivatized beads.

Standard histochemical analysis of cells and tissues generally involves procedures that utilize a relatively small number of probes such as dyes, and generally requires hours or days to process. Our laboratory has developed a novel method for histochemical surveys of cell surface properties that utilizes a large number of probes (derivatized agarose beads) and takes seconds or minutes to accomplish. In this study, 4 human cell lines (CCL-255 (LS123) human colon cancer cells that are non-tumorigenic in nude mice; CRL-1459 (CCD-18CO) human colon endothelial cells that are non-malignant; CCL-220 (COLO 320DM) human colon cancer cells that are tumorigenic in nude mice; and HTB-171 (NCI H446) human lung carcinoma cells) were tested for their ability to bind to agarose beads derivatized with 51 different molecules. There were statistically significant differences in binding of the 4 cell types to all of the 51 types of beads, but 15 types of beads showed dramatic differences in binding to one or more of the 4 cell types. For example, only HTB-171 (NCI H446) bound to p-aminophenyl-beta-D-glucopyranoside-derivatized beads and only CCL-220 (COLO 320DM) bound to L-tyrosine-derivatized beads. The specificity of cell-bead binding was examined by performing assays in the presence or absence of exogenously added compounds in hapten-type of inhibition experiments. This assay, that utilizes large numbers of novel probes, may help in the development of new libraries of surface properties of specific cell types, with differing degrees of malignancy, that at this time could not be developed by using other available technologies.

A new histochemical approach for studying sperm cell surfaces.

This study describes a novel rapid histochemical method to survey surface properties of sperm in a model system, the sea urchin. Surface properties of live and fixed sea urchin sperm of 2 species (Strongylocentrotus purpuratus and Lytechinus pictus) in seawater (live and fixed) and distilled water (fixed) were surveyed by assessing the ability of the sperm to bind to agarose beads derivatized with over 100 lectins and other proteins, carbohydrates, amino acids, nucleotides and other molecules. The results indicate that very little cell-bead binding occurs in seawater, whereas a great deal of binding occurs in distilled water, suggesting that ions inhibit cell-bead interaction. Whereas sperm of both species bound to many of the same beads, there were substantial differences in binding of sperm of each species to some beads, and there were differences in sperm-bead binding at different times of the year. The results suggest that the bead-binding assay enables the identification of species-specific and seasonally variable cell- surface properties. The large number of probes (bead types) available in the bead assay increases the likelihood of identifying new cell surface markers whose functional significance can then be the subject of further investigation. This assay offers an entirely new approach to examine surfaces of mammalian sperm in studies that could lead to identification of receptors involved in sperm-egg interaction, that may be candidates for the development of new contraceptive strategies.