Simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation using 7-amino-actinomycin D and pyronin Y.

We developed an improved technique that permits simultaneous DNA and RNA quantitation by a flow cytofluorometry using 7-amino-actinomycin D (7AAD) and pyronin Y (PY), respectively. Detailed cell cycle analyses based upon the cellular DNA/RNA levels were performed using cells suspended in a buffer containing 0.004% saponin. This method preserved the light scattering properties of human peripheral blood cells, thus lymphocyte, monocyte and granulocyte populations could be evaluated. In addition, since 7AAD and PY exhibit red (> 650 nm) and orange fluorescence (570 nm) respectively, the green fluorescence channel of the flow cytometer was reserved for surface phenotyping using FITC-conjugated antibodies. The 7AAD/PY method is applicable to the simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation when combined with FITC-conjugated surface markers in heterogeneous samples. To demonstrate the three-color analysis, PHA-activated human peripheral blood lymphocytes were stained for cell surface markers with monoclonal antibodies. The cells were suspended in buffer containing 0.004% saponin, then stained with 7AAD and PY. The DNA and RNA were analyzed in indivisual CD4+, CD8+ and CD20+ cells, and the characteristic cell cycle status was found. Cell activation was further analyzed using antibodies against interleukin-2 (IL-2) receptors (CD25), transferrin receptors (CD71) or HLA-DR molecules. Transferrin receptors were expressed in late G1 phase (G1B) just before the initiation of DNA synthesis, whereas IL-2 receptors and HLA-DR were expressed very early in the G1 phase (G1T). Since this technique preserves both light scatter properties as well as cell surface proteins, it is ideally suited for detailed cell cycle analyses of heterogeneous samples such as peripheral blood or bone marrow cells.