Combined Intracellular and Surface Staining

Detection of intracellular proteins by immunofluorescence allows one to determine the frequency and the light scatter and surface immuno-phenotype of protein-producing cells, irrespective of whether the protein is to be secreted, membrane bound, or localized in the cytoplasm. This is the only way to analyze secreted, cytoplasmic, and nuclear proteins by flow cytometry. Unfortunately, to date no method is known for cytoplasmatic immunofluorescence of live cells. In order to allow staining antibodies to penetrate the cell membrane, cells must be fixed and the membranes permeabilized. The choice of fixation method depends on the protein and its intracellular location and on the further use of the cells to be analyzed. For several applications fixation in formaldehyde and permeabilization of cell membranes by saponin (Willingham, M.C. and I. Pastan 1985) has been used successfully, including assessment of cytokines (Sander et al. 1991, Schmitz et al., 1992, in press). Formaldehyde is a cross-linking fixative with good preservation of cell morphology (Williams and Chase 1976; see figure, below). The plant glycoside saponin, a mild nonionic detergent, complexes with membrane cholesterol and other unconjugated s-hydroxysterols leading to ring-shaped complexes with a central pore about 8 nm in diameter (Bangham and Home 1962; Glauert et al. 1962). These pores allow passage of molecules of up to 200 kDa, (Schulz 1990). Since saponin acts in a reversible way (Willingham and Pastan 1985), it must be present in all incubation and washing steps; however, it allows surface staining at any point of the procedure if removed from the staining solution.