PMID: 7540639Jun 9, 1995Paper

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

Journal of Immunological Methods
K TobaA Shibata

Abstract

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 charac...Continue Reading

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Citations

Feb 13, 2001·Biochemical and Biophysical Research Communications·T NemotoS Kubota
Oct 10, 2002·Biochemical and Biophysical Research Communications·Tze HsiehJoseph Wu
Jan 17, 2002·Journal of Immunological Methods·Willem E CorverCees J Cornelisse
May 1, 2004·AIDS Research and Human Retroviruses·William J SwiggardMichael H Malim
Jul 22, 2006·Proceedings of the National Academy of Sciences of the United States of America·Hong-Hsing LiuZhijian J Chen
Oct 17, 2006·Cytometry. Part B, Clinical Cytometry·Zuhal HamurcuHamiyet Donmez-Altuntas
Jul 11, 2012·European Journal of Immunology·Hiromi ImamichiH Clifford Lane
Dec 22, 2009·Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photobiology·Pedro Montes-NavajasHermenegildo García
Nov 28, 2000·European Journal of Immunology·Q S LiY Komada
Mar 5, 2005·International Immunology·Hideya IgarashiPaul W Kincade
Sep 5, 2008·Current Protocols in Cytometry·Martin Poot
Sep 5, 2008·Current Protocols in Cytometry·I Schmid
Feb 5, 2000·The Journal of Immunology : Official Journal of the American Association of Immunologists·C WeiI Goldschneider
Sep 10, 2003·The Journal of Immunology : Official Journal of the American Association of Immunologists·Ronald L RabinJoshua M Farber

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