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.
BACKGROUND
The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle.


METHODS
Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry.


RESULTS
Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin).


CONCLUSIONS
The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.
Department of Pathology, University of Wales College of Medicine, Cardiff, United Kingdom. smithpj2@cf.ac.uk
P J
PJ
Smith
N
N
Blunt
M
M
Wiltshire
T
T
Hoy
P
P
Teesdale-Spittle
M R
MR
Craven
J V
JV
Watson
W B
WB
Amos
R J
RJ
Errington
L H
LH
Patterson
D000880:Anthraquinones; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000032:analysis*; D003952:Diagnostic Imaging / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007259:Infrared Rays; D008164:Luminescent Proteins / Q000378:metabolism; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D015394:Molecular Structure; D009589:Nitrogen Oxides; D015363:Quinolones / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D014105:Tosyl Compounds / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000880:Anthraquinones; C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D015342:DNA Probes; D004273:DNA, Neoplasm; C406029:DRAQ5NO; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009589:Nitrogen Oxides; D015363:Quinolones; D014105:Tosyl Compounds; D049452:Green Fluorescent Proteins; D016203:CDC2 Protein Kinase; C084731:zinquin