A direct hemolytic plaque assay on polylysine-coated coverslips for scanning electron microscopy of antibody forming cells.

A direct immunoplaque assay was utilized without agar or other semi-solid matrix for examining antibody secreting cells by scanning electron microscopy. For this purpose a polylysine procedure was used whereby 'charged' coverslips absorbed onto their surface an even monolayer of target sheep erythrocytes. Hemolysin secreting splenocytes derived either from mice immunized in vivo with SRBC or from normal or primed splenocytes immunized in vitro with the same antigen formed large numbers of hemolytic plaques on the RBC-coated polylysine treated coverslips. SEM examination of such plaque-forming cells revealed the presence of cells with the typical surface morphology of lymphocytes present in whole spleen cell suspensions. Many of the lymphocytes were covered with numerous microvilli while a small percentage were relatively free of such villi and exhibited a much smoother surface. Examination of lymphocytes secreting hemolytic antibody on polylysine coated coverslips sensitized with target erythrocytes thus permits the direct examination and analysis of surface morphologic features and also permits a direct comparison with other features revealed by light and fluorescent microscopy.