Use of SYTO 13, a fluorescent dye binding nucleic acids, for the detection of microparticles in in vitro systems.
Microparticles (MPs) are small membrane-bound vesicles that are released from activated or dying cells by a blebbing process. These particles contain nuclear and cytoplasmic components and represent unique biomarkers for disease. The small size of particles, however, limits detection using flow cytometry with either light scatter or staining for surface markers. Because MPs contain DNA and RNA, we have explored the use of SYTO 13, a member of the class of SYTO dyes, for particle detection. SYTO 13 is cell permeable and has a high fluorescent yield when bound to DNA or RNA. In this study, we compared detection of MPs using either light scatter or SYTO 13 staining, testing the hypothesis that, with fluorescence detection with SYTO 13, problems of "noise" with light scatter are reduced and the range of MP sizes detected is increased. In these experiments, particles were obtained from lymphoid cell lines treated in vitro to undergo apoptosis. As these results showed, STYO 13 allowed the detection of 1.5-2.9 times as many particles as did light scatter. The increased sensitivity was observed with three different cell lines and was independent of inducing stimulus. Treatment of fixed and permeabilized MPs with DNase and RNase showed that SYTO 13 binding resulted from interaction with both DNA and RNA. Together, these findings indicate that the nucleic acid content of MPs provides the basis for their detection in in vitro systems and suggests the utility of fluorescent dyes like SYTO 13 for more sensitive quantitative assays.
Ullal
Anirudh J
AJ
Division of Rheumatology and Immunology, Department of Medicine, Duke University, Durham, North Carolina, USA. anirudh.ullal@duke.edu
Pisetsky
David S
DS
Reich
Charles F
CF
D017209:Apoptosis; D004247:DNA / Q000737:chemistry; D003851:Deoxyribonucleases / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D019169:Jurkat Cells; D008027:Light; D008863:Microspheres; D009696:Nucleic Acids / Q000737:chemistry*; D009930:Organic Chemicals / Q000494:pharmacology; D012313:RNA / Q000737:chemistry; D012260:Ribonucleases / Q000378:metabolism; D012542:Scattering, Radiation
D005456:Fluorescent Dyes; D009696:Nucleic Acids; D009930:Organic Chemicals; C461159:SYTO 13; D012313:RNA; D004247:DNA; D003851:Deoxyribonucleases; D012260:Ribonucleases