RESUMEN
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.
Asunto(s)
Membrana Celular/metabolismo , Histocitoquímica , Microesferas , Receptores de Quimiocina , Animales , Arginina/farmacología , Quimiocina CCL20 , Quimiocinas CC/química , Quimiocinas CC/metabolismo , Concanavalina A/farmacología , Histidina/química , Humanos , Proteínas Inflamatorias de Macrófagos/química , Proteínas Inflamatorias de Macrófagos/metabolismo , Ratones , Ratones Desnudos , Receptores CCR6 , Sefarosa/química , Células Tumorales CultivadasRESUMEN
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.