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

INTRODUCTION

Flow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G₀ phase cannot be discriminated from cells in the proliferative G₁ 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 G₀ and cell-cycle subcompartments of G₁. 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.