Flow cytometric immunofluorescence assay for quantification of cyclobutyldithymine dimers in separate phases of the cell cycle.

Abstract

Quantitative immunofluorescence assays for the measurement of cyclobutyldithymine dimers (T <> T) based on computer-assisted immunofluorescence microscopy have recently been described. Here we present a modified assay for T <> T based on flow cytometry. This method has the advantage that T <> T can be quantified in separate phases of the cell cycle by the fluorescent counterstaining of nuclear DNA and subsequent selection on DNA content. The H3 monoclonal antibody directed at T <> T binds to partially denatured DNA in situ. The antibody is labeled with fluorescein isothiocyanate (FITC) and DNA is stained with the intercalating dye 7-amino-actinomycin D. FITC fluorescence increases linearly with dose of UV-C radiation (up to 45 J/m2) of cultured human fibroblasts. A linear fluorescence-dose relationship was also found for epidermal cells of SKH:HR1 hairless mice after in vivo irradiation with UV-B (FS40 sunlamp, up to 3750 J/m2). This technique allows a quick assessment of UV damage levels in 10,000s of cells and makes immunofluorescence of DNA damage more accessible to other research groups.