Quantification of an In Vitro Cell - Cell Adhesion Assay Using Interactive Laser Scanning Cytometry
We are interested in identifying cellcell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cellspermatogenic cell adhesion. Numerous cell‐cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225—500 μm2, spermatids were 100—225 μm2 and residual bodies were < 100 μm2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub‐classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell‐cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell‐cell adhesion assay in which one cell type is adherent to a substrate.
cell‐cell adhesion assay, sertoli cell, spermatogenic cell, scanning laser cytometry
Newton, Sean C., and Clarke F. Millette. "Quantification of an in vitro cell‐cell adhesion assay using interactive laser scanning cytometry." Cytometry: The Journal of the International Society for Analytical Cytology 13, no. 3 (1992): 209-219.