DRAQ5™ far-red fluorescent DNA dye

DRAQ5™ far-red fluorescent DNA dye is a novel cellular imaging reagent that can be used in live cells or fixed cells in combination with other common fluors. DRAQ5™ emits in the far-red (665 nm and beyond), making it ideal for use with GFP and all visible range-fluor tagged ligands. Due to this spectral separation, the GFP and nuclear signals can be captured simultaneously. For this reason alone, the screening time is HALVED!!

DRAQ5 directly and stoichiometrically reports DNA content in cells, live or fixed.  Stained cells can be gated via flow cytometry and sorted into isolated populations with different DNA content relating to the cell cycle, such as elevated G2/M or aneuploidy.  The DNA from the cell fractions can then be used directly for population-restricted PCR amplification.  Because DRAQ5 is a far-red emitting DNA dye this can be easily multiplexed with other parameters, like cell surface phenotype.

DRAQ5™ differentially stains both the nucleus and cytoplasm making it ideal for any HCS translocation (or Redistribution™ / Transfluor™) assay.  The intensity thresholding for the two compartments improves an in silico “dilation” while providing morphometrics which can be used to indicate toxicity.  DRAQ5™ has also been successfully used in HCS assays (e.g. GPCRs) measuring cell cycle, ploidy, or protein expression to gain speed, improve performance and efficiency.

Applications:
HTS / HCS – Nuclear Visualization; Translocation Assays; PE Opera Assays
Confocal Microscopy – Live Cell imaging; IHC/IF; Fixed Cells/Nuclei
Flow Cytometry – Flow Sorting Cells for PCR; Cell Cycle Analysis; Cell Ploidy
Laser Scanning Slide & Microplate Cytometry – Cell Cycle Analysis on LSC; Microplate Cytometry
Lamp/Camera Microscopy

Comparison Chart of Nuclear DNA Dyes for Live Cell Imaging

Spectral Profile
http://www.biostatus.com/product/draq5/spectral_profile/

 Key References

• Smith PJ. Blunt N. Wiltshire M. Hoy T. Teesdale-Spittle P. Craven MR. Watson JV. Amos WB. Errington RJ. Patterson LH. 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(4):280-91, 2000.
• Wiltshire M. Patterson LH. Smith PJ. A novel deep red/low infrared fluorescent flow cytometric probe, DRAQ5NO™, for the discrimination of intact nucleated cells in apoptotic cell populations. Cytometry. 39(3):217-23, 2000.
• Smith PJ. Wiltshire M. Davies S. Patterson LH. Hoy T. A novel cell permeant and far red-fluorescing DNA probe, DRAQ5™, for blood cell discrimination by flow cytometry. Journal of Immunological Methods. 229(1-2):131-9, 1999

DRAQ™ is a trademark of Biostatus Ltd. DRAQ5™ is intended for research purposes only.

Sample Protocol:

LABELING INTACT LIVE CELLS:

• Cells can be stained directly without any fixation or permeabilization
• Just a few minutes (and no washing) cells are ready for imaging (protocols)
• Add it to the assay medium in a live cell assay (e.g. GPCR ligands)

FIXED CELLS & NUCLEI

• Fix and wash your cells then just add DRAQ5™ in the normal way
• more information and examples

See Biostatus's website for more details: protocols

The 50μl vial (DR50050) will give you:
150 Flow Cytometry Assays
250 Imaging Assays

Typical uses of the 200uL DRAQ5 vial (DR50200):
600 assays by flow cytometry
1000 confocal imaging assays
2000 assays in a HTS/HCS screen

DRAQ™ is a trademark of Biostatus Ltd. DRAQ5™ is intended for research purposes only.

A selection of publications where DRAQ5 has been described for high content screening:

• DRAQ5 HCS MINI-REVIEW
• In GE Healthcare Biosciences' IN Cell™ AKT1-GFP Assay protocol DRAQ5™ halves the assay scan time for a few cents per well, surely a very small price for such an improvement. Link to the user manual which describes the advantage of DRAQ5™'s emission not overlapping with that of GFP cf. Hoechst dye.
• High Content Screening for G Protein-Coupled Receptors Using Cell-Based Protein Translocation Assays. Grånäs C, Lundholt BK, Heydorn A, Linde V, Pedersen H-C, Krog-Jensen C, Rosenkilde MM, Pagliaro L. Combinatorial Chemistry & High Throughput Screening 2005; 8: 301-309. BioImage A/S, Denmark & The Panum Institute, Denmark
• Development and Validation of Algorithms for Measuring G-Protein Coupled Receptor Activation in Cells Using the LSC-Based Imaging Cytometer Platform. Ozawa K,Hudson CC, Wille KR, Karaki S, Oakley RH. Cytometry Part A 2005; 65A:69–76. Olympus Corp., Japan & Xsira Pharmaceuticals, USA.
• Screening for GPCR agonists and antagonists with ImageXpressMICRO, the Transfluor assay and AcuityXpress. Paula Rickert, Molecular Devices
  Corp., USA; P8-9, The Molecular Messenger December 2005.
• Cytomics – Drug Discovery via Cytometry in Time and Space. Paul J Smith, Univ. of Wales College of Medicine, UK; P40-44, BusinessBriefing: Pharmatech 2004
• Screening of signaling events in live cells using novel GFP redistribution Assays. Stubbs, et al. Amersham Biosciences, UK & BioImage AS, Denmark; Poster, IBC Conference 2001.

 
A selection of publications where DRAQ5 has been described for live cell imaging:

• Foley et al. Frontiers in Bioscience 2005; 10:1302-1312
  Live cell imaging of glial cells & VSMCs was required as both ethanol and PFA fixation generated unwanted artefacts. DRAQ5 was completely compatible with the fluorescein-labelled peptide.
• Schjetne et al. The Journal of Immunology 2003; 171:32–36
  Live cell imaging was required as they were observing the internalisation of an (Alexa 546) antibody to TLR-2 into cells transfected with GFP-tagged marker of endosomes. DRAQ5, GFP and Alexa 546 were spectrally compatible.
• See a review of a paper by Martin et al (2005) for an independent view
• See the comparison chart of different live cell dyes based on this paper

Conditions: 3000-4000 cells in one 384 well. Cells were fixed with PFA and stained with 20µL of 2.5µM DRAQ5™ in PBS for 30 min at RT.

Image acquisition settings: standard Evotec Opera™ QEHS settings for 2 colours, 20x objective. Parallel images from the target and DRAQ5™ channels with excitations at 488 and 635nm: primary dichro 488/635, detection green: 535/60 (60), detection red: 690/50. The green GFP fluorescence was quantified with respect to each cell (DRAQ5 stain).

Biostatus is grateful to Drs. Andreas Scheel and Stefan Jaeger, Evotec AG, Hamburg for providing these images.

"Could DRAQ5 be used to label dsDNA or RNA itself? I would like to measure how cells are taking up DNA and RNA."

DRAQ5™ labels dsDNA. It intercalates DNA at the A-T sites and will do so stoichiometrically.

Although DRAQ5 probably does label some RNA, in practice this is not seen – mainly due to the very low quantum yield of DRAQ5 and the distributed presence of RNA in a cell. So for all intents and purposes, DRAQ5 can be considered as a dsDNA dye.

 "What size do I need? How many assays will the 50μl and 200μl vials provide?"

The 50μl vial (DR50050) will give you:
150 Flow Cytometry Assays
250 Imaging Assays

Typical uses of the 200uL DRAQ5 vial (DR50200):
600 assays by flow cytometry
1000 confocal imaging assays
2000 assays in a HTS/HCS screen

"May I use DRAQ5 as a replacement for Hoechst to look at apoptotic nuclei?"

DRAQ5 can replace Hoechst stain for staining live or fixed cells. With its far-red excitation/emission, there is no need to expose cell to UV light.

Whereas it is possible to gain information on nuclear morphology with the DRAQ5 dye, for apoptotic differentiation, we would recommend using the DRAQ7™ dye.  This vital dye does not penetrate living cells, so it will only stain dead and membrane-compromised cells. The catalog number of DRAQ7 is DR71000.

Call 1-800-829-3194 for technical assistance or email Technical Support: help {at} immunochemistry.com.

DRAQ™ is a trademark of Biostatus Ltd. DRAQ5™ is intended for research purposes only.