Open AccessA method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry
Author(s) -
Clevenger Charles V.,
Bauer Kenneth D.,
Epstein Alan L.
Publication year - 1985
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990060306
Subject(s) - flow cytometry , microbiology and biotechnology , monoclonal antibody , immunofluorescence , paraformaldehyde , antigen , propidium iodide , staining , biology , cell cycle , cytometry , antibody , cell , chemistry , biochemistry , immunology , apoptosis , genetics , organic chemistry , programmed cell death
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°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.