DRAQ5™ (ab108410)

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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.

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.

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.

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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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.