Overview

  • Product name
    DRAQ5™
  • Description
    DRAQ5™
  • Tested applications
    Suitable for: FM, Flow Cyt, ICC/IFmore details
  • General notes

    DRAQ5™ is a cell permeable far-red fluorescent DNA dye that can be used in fixed or non-fixed/ live cells in combination with common labels such as GFP or FITC.

    As with any cell­-permeant DNA intercalating probe, DRAQ5 may inhibit cell division in long-term assays and should be tested for any effect.

    DRAQ5 staining can be used in flow cytometry, live cell imaging and cell-based assays and the dye is highly compatible with standard protocols across many instrumentation platforms.

    The chemical name of DRAQ5 is 1, 5-bis{[2-(di-methylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9, 10-dione.

    The advantages of DRAQ5 staining include
    - convenient ready-to-use aqueous solution
    - rapid uptake into living cells, providing a high level of nuclear discrimination
    - no photobleaching effect
    - can be used in most cell types, eukaryotic and prokaryotic: mammalian, bacterial, parasitic, plant, etc.
    - no compensation needed with common FITC/GFP + PE combinations in flow cytometry
    - no RNase treatment required
    - no fluorescence enhancement upon DNA binding
    - compatible with optics of benchtop flow, laser scanning cytometers and non-UV laser scanning and lamp-based confocal microscopes

    SPECTRAL PROPERTIES:

    Excitation

    • 647 nm line optimal (Exmax 646 nm)
    • 488, 514, 568 and 633 nm lines, sub-optimal
    • Two-photon excitation (1047 nm) and excitation dark (700-850 nm)

    Emission (instrument dependent):
    - 665 nm to infra-red max 681 nm / 697 nm intercalated with dsDNA)
    - minimal overlap with vis range e.g. GFP and FITC
    - Em. filters may include 695L, 715LP or 780 LP

     

     

    Concentration: 5 mM

Properties

Applications

Our Abpromise guarantee covers the use of ab108410 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
FM Use at an assay dependent concentration.
Flow Cyt 1/1000 - 1/250. For Gating of Nucleated cells = 5µM; For Cell Cycle Analysis = 20µM
ICC/IF 1/1000. For Cell-based assays, Immunofluorescence microscopy and In-Cell WB = 5µM
It is highly recommended that the concentration and labelling conditions are carefully determined by each investigator for optimal performance in the assay of interest. For more specific information about the applications, please refer to the Protocol Booklet section.

Images

References

This product has been referenced in:
  • Lawlor KT  et al. Nephron progenitor commitment is a stochastic process influenced by cell migration. Elife 8:N/A (2019). Read more (PubMed: 30676318) »
  • Cheedipudi SM  et al. Genomic Reorganization of Lamin-Associated Domains in Cardiac Myocytes Is Associated With Differential Gene Expression and DNA Methylation in Human Dilated Cardiomyopathy. Circ Res 124:1198-1213 (2019). Read more (PubMed: 30739589) »
See all 32 Publications for this product

Customer reviews and Q&As

1-10 of 37 Abreviews or Q&A

Answer

Thank you for contacting us.

The information given in page 7 of protocol booklet states "DRAQ5™ staining is accelerated at 37°C and incubation time (5-30 minutes) may be reduced but this should be checked by titration and for each cell type. DRAQ5™ may be added to the assay medium for the duration of the assay (typically 0.5 – 3 hours) at 1µM prior to any agonist / antagonist additions.

DRAQ5™ binds irreversibly to the cellular DNA which is ultimately cytotoxic to cells. DRAQ5™ staining occurs very quickly, so we suggest adding DRAQ5™ to live culture cells just before analysis. Cells should not be incubated longer than 3 hours however it may vary with cell line.

For longer term viability assays, we recommend DRAQ7™ (ab109202).

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your email. I am sorry for the inconvenience. Please check the following DRAQ5 specific publications;
http://www.ncbi.nlm.nih.gov/pubmed/14994375
http://www.cbm.uam.es/mkfactory.esdomain/webs/cbmso/images/TextBox/DRAQ5.pdf
http://www.pathology.ufl.edu/˜lyang/index_files/Publications/Yuan%20C%202004%20FC.pdf
http://www.sciencedirect.com/science/article/pii/S0022175999001167
Other general references are
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
I hope this information will be helpful.

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DRAQ5: Cell cycle analysis with flow cytometry

Excellent Excellent 5/5 (Ease of Use)
Abreviews
DRAQ5 has been used with LNCaP C4-2B cell line in flow cytometry for cell cycle analysis. The cells were scraped and centrifuged for 5 min at 4C at 1.200 rpm. Washed with cold PBS and further centrifuged for 5 min at 4C at 1.200 rpm. The cells were then stained with 200 ul of staining solution (DRAQ5 1:1000 dil in PBS) for 1 h at room temperature protected from lighted. The reaction volume was finally made up to 500 ul and the cells were examined by flow cytometry. There is a distinct difference in the apoptotic induced treated cells with a high apoptoptic percentage (M1) and lower M2, M3 and M4 percentages, representing G1, S and G2-M phases, respectively.

Dr. Dimitra Kalamida

Verified customer

Submitted Aug 22 2018

DRAQ5 In cell Western As Part of an Ab Trial scheme

Excellent Excellent 5/5 (Ease of Use)
Abreviews
DRAQ5 has been used to normalize for cell number( C42B cell line) . DRAQ5, a DNA dye has been used to label nuclei, in 1:1000 dilution in PBS, with a total volume of 50µl/ well. The cells were incubated for 30 minutes at room temperature in dark, before scanning using appropriate excitation (648 nm) and emission (705LP) filters. Cell number titration has been performed using 60000, 30000, 15000 and 7500, respectively. The fluorescence intensity of each well was quantified using ImageJ and plotted in a graph using GraphPrism, as demonstrated in following figure .

Dr. Dimitra Kalamida

Verified customer

Submitted Jun 05 2018

So good!

Good Good 4/5 (Ease of Use)
Abreviews
It is easy for staining with DAPI! so good.

Ms. Sehee Oh

Verified customer

Submitted Jan 31 2018

Answer

Vielen Dank für Ihre Anfrage.


Ich kann bestätigen, dass die DRAQ5 Färbung bei höheren Temperaturen verstärkt wird und deshalb die Inkubationszeit vielleicht angepasst werden muss. Je länger die Inkubationszeit desto mehr Signal erwarten wir bis die DNA abgesättigt ist. Sollte DRAQ5 dann entzogen werden würden wir einen langsamen Rückgang der Färbung über Zeit erwarten.


DRAQ5 fluoresziert kaum wenn es nicht an dsDNA gebunden ist. Erst wenn es in dsDNA interkaliert ist gibt es ein starkes fluoreszentes Signal. Eine DRAQ5 Färbung ist in der Apoptose nicht beeinträchtigt da die Affinität des DRAQ5 zu dsDNA sich nicht in lebenden oder toten Zellen unterscheidet. Da sich in der Apoptose die Menge an dsDNA ändert wird sich auch die Stärke des DRAQ5 Signal ändern im Verhältnis zu der Veränderung der dsDNA.


Ich hoffe, diese Information ist hilfreich und wünsche Ihnen viel Erfolg bei Ihrer Forschung.

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Answer

1) We would expect the cell staining to diminish after a medium exchange. Despite DRAQ5’s high affinity for dsDNA there is still an equilibrium in place and this will be disturbed by removal of the low residual concentration in the culture medium.

2) I think the staining might persist for 1-2 h after the removal but this may depend upon the previously used concentration that the cells were exposed to. This is a paradox of course since one would want to keep the concentration low (perhaps 0.5 - 1 uM) if there’s any evidence of toxicity.

For sure, it is completely cell line and state dependent whether DRAQ5 will have a toxic impact. It would be reasonable to predict that a cell which is not in a proliferative state would be less susceptible to any cell permeant DNA intercalating probe. For example, it may be more likely to be OK with terminally-differentiated cell types like macrophages or HUVECs where it and the closely related CyTRAK Orange have been demonstrated in longer-term experiments.

If there has been no evidence of toxicity then I would continue for the 18hr experiment in the presence of DRAQ5.

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DRAQ5 co-staining

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Excellent
Compatible with FITC, Alexa 568, and Alexa 405 co-staining

Abcam user community

Verified customer

Submitted Dec 08 2014

DRAQ5 Abcam for In Cell Western Assay

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Our lab uses DRAQ5 from Abcam for the In Cell Western Assay. DRAQ5 is used to provide accurate normalization over a broad range of cell densities. Specifically, it can be used for stoichiometric staining of DNA in live or fixed cells. DRAQ5 is used in the 700nm channel to normalize well-to well variation in cell number.
Picture shows In Cell Wester Assay using DRAQ5 in PC3 and LnCAP prostate cell lines.

Abcam user community

Verified customer

Submitted Jun 25 2014

Answer

Thank you for contacting us.



1. The most important consideration would be keep the cells in the presence of DRAQ5 and not to wash them. The nuclear stain should persist well but the likelihood is that progressively there would be a small amount of staining of lipophilic structures but this not likely to have any impact on the discrimination of nucleated from non nucleated cells.



In some HCS assays DRAQ5 positive live cells have been monitored for many hours (2h) and I would expect for fixed cells, as for slide based imaging it would be many weeks. The issue with the live cells would be if they are proliferating as any DNA intercalating dye will impact on cell cycle / arrest. There is likely to be some exchange between the lumen of the sample buffer and the DNA bound DRAQ5 over time as the volume is great and cells will degrade.



2. It is difficult to be definitive about this as it may well be cell type specific. However, the affinity for dsDNA in live or dead cells in unlikely to be affected but the amount of dsDNA will almost certainly be.



3. So, it is possible to remove a sub-G1 population from the analysis but this will not account for late apoptotic cells or necrotic cells where DNA fragmentation has not occurred.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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1-10 of 37 Abreviews or Q&A

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