Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens

Fixation and immunofluorescent staining methods were developed for analyzing intracellular antigens with the cell flow cytometer. Fixing cell suspensions with 100% methanol provided best preservation of morphology, lowest fluorescent background staining and most intense specific immunofluorescence. Immunoglobulins present in B cell lines that were representative of different developmental stages could be distinguished quantitatively. Fluorescence histograms were compared with fluorescence microscope presentation of stained cells. Intracellular antigens that reacted with monoclonal antibodies could also be evaluated by flow cytometry. This method was utilized to assess plasmacyte development in mouse spleen cell cultures after stimulation with lipopolysaccharide.