Combined Flow Cytometric Measurement of Two Cell-Surface Antigens and DNA-RNA Content

INTRODUCTIONFlow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G(0) phase cannot be discriminated from cells in the proliferative G(1) phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G(0) and cell-cycle subcompartments of G(1). Assessing phenotype at the same time as nucleic acid content allows determination of the cell-cycle status of subpopulations in mixed-cell preparations. This protocol describes an optimized method for combining dual-color cell-surface immunofluorescent staining with staining for DNA-RNA, adapted for a basic dual-laser flow cytometer with blue (488-nm) and red (633- or 647-nm) excitation. DNA is stained at low pH in the presence of saponin with 7-aminoactinomycin D (7-AAD), and RNA is stained with pyronin Y (PY). Both dyes are used at low concentration, and 7-AAD is exchanged with nonfluorescent actinomycin D to minimize fluorochrome-fluorochrome interactions, which can negatively affect detection of cell-surface antigen staining.