A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry.
A preparative technique for the two-parameter flow cytometric study of nuclear antigen expression is reported. This method employs a brief sequential treatment of cells at 4 degrees C first with 0.5% paraformaldehyde and second with 0.1% Triton X-100 in phosphate-buffered saline followed by cellular staining with indirect immunofluorescence and propidium iodide. Using this technique, cellular morphology is preserved, cell clumping is minimized, and high-quality indirect immunofluorescence and DNA staining are obtained with a minimum of nonspecific labeling. Utilizing nuclear antigen-specific monoclonal antibodies in conjunction with this technique, the cell-cycle phase-dependent expression of such antigens is examined. From these data, the utility of two-parameter flow cytometry in the identification and quantification of cell-cycle-dependent modulation of nuclear antigens is discussed.
D000911:Antibodies, Monoclonal / Q000276:immunology; D034961:Antigens, Nuclear; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000276:immunology*; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde; D006801:Humans; D009698:Nucleoproteins / Q000032:analysis*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011108:Polymers; D011419:Propidium
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D009698:Nucleoproteins; D011108:Polymers; D005557:Formaldehyde; D011419:Propidium; D011092:Polyethylene Glycols; D017830:Octoxynol; D004247:DNA; C003043:paraform