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http://dx.doi.org/10.1007/s10059-009-0066-3

Use of DNA-Specific Anthraquinone Dyes to Directly Reveal Cytoplasmic and Nuclear Boundaries in Live and Fixed Cells  

Edward, Roy (Biostatus Ltd.)
Publication Information
Molecules and Cells / v.27, no.4, 2009 , pp. 391-396 More about this Journal
Abstract
Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events - for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5$^{TM}$ and CyTRAK Orange$^{TM}$. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.
Keywords
anthraquinones; cell-based assays; cytometry; DNA dyes; fluorescent; high content screening; in-cell western;
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1 Gustin, E., Van Loock, M., Van Acker, K., and Krausz, E. (2009). Weak signals and strong artifacts: HCS in antiviral drug discovery. Oral presentation: High Content Analysis Conference, USA, p. 5
2 Loechel, F., Bjorn, S., Linde, V., Praestegaard, M., and Pagliaro, L. (2006). High content translocation assays for pathway profiling. In Methods in Molecular Biology, Vol. 356: High content screening: a powerful approach to systems cell biology and drug discovery. D.L. Taylor, J.R. Haskins, and K. Giuliano, eds. (Totowa, USA: Humana Press, Inc.), pp. 401-414
3 Plander, M., Brockhoff, G., Barlage, S., Schwarz, S., Rothe, G., and Knuechel, R. (2003). Optimization of three- and four-color multiparameter DNA analysis in lymphoma specimens. Cytometry 54A, 66-74   DOI   PUBMED
4 Smith, P.J., Blunt, N., Wiltshire, M., Hoy, T., Teesdale-Spittle, P., Craven, M.R., Watson, J.V., Amos, W.B., Errington, R.J., and Patterson, L.H. (2000). Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy. Cytometry 40, 280-291   DOI   ScienceOn
5 Xu, J.J., Henstock, P.V., Dunn, M.C., Smith, A.R., Chabot, J.R., and de Graaf, D. (2008). Cellular imaging predictions of clinical druginduced liver injury. Toxicol. Sci. 105, 97-105   DOI   ScienceOn
6 Visconti, R.P., Ebihara, Y., LaRue, A.C., Fleming, P.A., McQuinn, T.C., Masuya, M., Minamiguchi, H., Markwald, R.R., Ogawa, M., and Drake, C.J. (2006). An in vivo analysis of hematopoietic stem cell potential hematopoietic origin of cardiac valve interstitial cells. Circ. Res. 98, 690-696   DOI   ScienceOn
7 Yuan, C.M., Douglas-Nikitin, V.K., Ahrens, K.P., Luchetta, G.R., Braylan, R.C., and Yang, L. (2004). DRAQ5-based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties. Cytometry 58, 4752   DOI
8 Payne, S., Wylie, P., Edward, R., and Goulter, A. (2007). Use of farred emitting DNA dye DRAQ5 for cell cycle analysis with microplate cytometry. Poster Presentation: Drug Discovery and Technology Conference, USA
9 Hannoush, R.N. (2008). Kinetics of Wnt-driven b-Catenin stabilization revealed by quantitative and temporal imaging. PLoS ONE 3, e3498   DOI   PUBMED   ScienceOn
10 Swerts, K., Van Roy, N., Benoit, Y., Laureys, G., and Philippe, J. (2007). DRAQ5: Improved flow cytometric DNA content analysis and minimal residual disease detection in childhood malignancies. Clin. Chim. Acta 379, 154-157   DOI   PUBMED   ScienceOn
11 Martin, R.M., Leonhardt, H., and Cardoso, M.C. (2005). DNA labeling in living cells. Cytometry 67A, 45-52   DOI   PUBMED   ScienceOn
12 Primo, D., Flores, J., Quijano, S., Sanchez, M.L., Sarasquete, M.E., del Pino-Montes, J., Gaarder, P.I., Gonzalez, M., and Orfao, A. (2006). Impact of BCR/ABL gene expression on the proliferative rate of different subpopulations of haematopoietic cells in chronic myeloid leukaemia. Brit. J. Haematol. 135, 43-51   DOI   PUBMED   ScienceOn
13 Bjornsson, S., Wahlstrom, S., Norstrom, E., Bernevi, I., O'Neill, U., Johansson, E., Runstrom, H., and Simonsson, P. (2008). Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer. Cytometry 74, 91-103   DOI   PUBMED   ScienceOn
14 Richardson, C., et al. (2008). Development of a mechanistic cellbased assay for the identification of JAK2 inhibitors. Poster presentation: SBS/ELRIG drug discovery conference, UK
15 Pelkmans, L., Fava, E., Grabner, H., Hannus, M., Habermann, B., Krausz, E., and Zerial, M. (2005). Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436, 78-86   DOI   PUBMED   ScienceOn
16 Foley, K.F., De Frutos, S., Laskovski, K.E., Tegge, W., and Dostmann, W.R. (2005). Culture conditions influence uptake and intracellular localization of the membrane permeable cGMP-dependent protein kinase inhibitor DT-2. Front. Biosci. 10, 1302-1312   DOI   PUBMED
17 Garcia-Escarp, M., Martinez-Munoz, V., Sales-Pardo, I., Barquinero, J., Domingo, J.C., Marin, P., and Petriz, J. (2004). Flow cytometrybased approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs. Cytometry 62A, 129-138   DOI   PUBMED
18 Amersham Biosciences (2003). GFP-Rac1 assay: user manual, section 5.2.7. 25-8007-27UM, Rev. A
19 Rosado, A., Zanella, F., Garcia, B., Carnero, A., and Link, W. (2008). A dual-color fluorescence-based platform to identify selective inhibitors of Akt signaling. PLoS ONE 3, e1823   DOI   ScienceOn
20 Cogger, V.C., Arias, I.M., Warren, A., McMahon, A.C., Kiss, D.L., Avery, V.M., and Le Couteur, D.G. (2008). The response of fenestrations, actin, and caveolin-1 to vascular endothelial growth factor in SK Hep1 cells. Am. J. Physiol. Gastrointest. Liver Physiol. 295, G137-G145   DOI   PUBMED   ScienceOn
21 Errington, R.J., Pors, K., Patterson, L.H., Ogrodzinski, S.J., and Smith, P.J. (2006). $CyTRAK^{TM}$ probes: novel nuclear and cytoplasm discriminators compatible with GFP-based HCS and HTS assays. Poster presentation: Society for Biomolecular Sciences Annual Conference, USA
22 Simonen, M., Ibig-Rehm, Y., Hofmann, G., Zimmermann, J., Albrecht, G., Magnier, M., Heidinger, V., and Gabriel, D. (2008). High-content assay to study protein prenylation. J. Biomol. Screen. 13, 456-467   DOI   PUBMED
23 Conour, J.E., Graham, W.V., and Gaskins, H.R. (2004). A combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression. Physiol. Genom. 18, 196-205   DOI   PUBMED   ScienceOn
24 George, T.C., Morrissey, P.J., Cui, C., Singh, S., and Fitzgerald-Bocarsly, P. (2008). Kinetics of CpG internalization and sub-cellular organelle co-localization within circulating human plasmcytoid dendritic cells. Application note, Amnis Corp
25 May, K., Preckel, H., Schaaf, S., and Mumtsidu, E. (2008). Imagebased quantification of cyclin B1 and DNA content during cell cycle using the opera HCS platform. Poster Presentation: SBS/ELRIG Drug Discovery Conference: UK
26 Haasen, D., Wolff, M., Valler, M.J., and Heilker, R. (2006). Comparison of G-Protein coupled receptor desensitization-related $\beta$arrestin redistribution using confocal and non-confocal imaging. Comb. Chem. High Throughput Screen. 9, 37-47   DOI   ScienceOn
27 Laakkonen, J.P., Kaikkonen, M.U., Ronkainen, P.H.A., Ihalainen, T.O., Niskanen, E.A., Hakkinen, M., Salminen, M., Kulomaa, M.S., Yla-Herttuala, S., Airenne, K.J., et al. (2007). Baculovirus-mediated immediate-early gene expression and nuclear reorganization in human cells. Cell. Microbiol. 10, 667-681   DOI   PUBMED   ScienceOn