This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4

Simultaneous analysis of DNA content and surface immunophenotype using gentle ethanol fixation techniques.

William Telford. Louis E. King and Pamela J. Fraker
Michian State University, East Lansing, MI
Hospital for Special Surgery, New York, NY

A major issue in the simultaneous analysis of DNA content using DNA binding dyes and cell surface immunophenotyping using fluorochrome-conjugated antibodies is the choice of cell pretreatment technique. For successful DNA content measurement, cell and nuclear membranes need to be sufficiently permeablized to allow entry of the DNA binding dye. Ethanol or other alcohols have been traditionally used to permeablize cells for DNA content analysis; this treatment, however, has the disadvantage of damaging cell surface proteins, as well as causing dissociation of fluorochrome-conjugated antibodies from cell surface antigens. Crosslinking of cell surface proteins with paraformaldehyde, either by itself or prior to ethanol treatment, has been previously used in an attempt to circumvent this problem. Paraformaldehyde crosslinks chromatin, however, limiting stoichiometric binding of DNA probes and reducing the quality of the resulting DNA content distribution. In an attempt to accurately measure DNA content with simultaneous preservation of cell surface markers, we have utilized gentle ethanol treatment techniques, which permeablize cells with minimal loss of surface antigen expression and antibody-antigen association. For some cell types, the presence of apoptotic cells based on reduced DNA content can also be detected. One such technique employs the addition of ethanol to cells previously resuspended in high concentrations of fetal bovine serum, and has been thoroughly described in the literature. A second technique uses ethanol solutions in glycerol, and is still under development.


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