Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y

Cytometry
H M Shapiro

Abstract

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 ...Continue Reading

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