DRAQ7™ (ab109202)

Ich habe nun vom Labor eine Antwort erhalten und kopiere diese im Original für Sie:

"In brief, it is likely that there will be significant washout of the DRAQ7 from the leaky cells into the previously viable ones when performing the multiple steps of subsequent IF staining, that is, after fixation.

This does not happen when the endpoint is just fixation with Formaldehyde and retaining this in the sample at 1% preserves the differential between leaky and intact cells (eg with a FP reporter and/or Hoechst for all cells), and good for extended periods ( >24 h)."

Zusammenfassend können wir DRAQ7 nicht empfehlen, wenn nach der Fixierung noch weiter Färbeschritte durchgeführt werden.

The bottom line is that toxicity studies have yet to been done on DAPI. I have seen one article questioning its use (Qin, 2011 using it on MSCs).
DAPI is known to be semi-permeant to cells and thus it does get in. As a very high affinity DNA binding dye with a degree of permeance it will almost certainly have toxic effects. This is the nature of DNA-intercalators.

To the contrary, DRAQ7 has already been tested in highly sensitive bioassays for DNA damage (see Akagi et al, Feb 2013 issue of Cytometry Part A) and even after several days at very high concentrations did not have an impact on cell health. They also used DRAQ7 alongside TMRM as a reporter of mitochondrial health and the read-outs are equally supportive of DRAQ7 as a long-term, real-time reporter of cell integrity / permeabilisation.

We have other anecdotal evidence in rapidly proliferating SUD-4-DHL cells over 4 days and a further collaborator has been using it in real-time analysis of nano-particle toxicity in fibroblast cultures, over 2 days.

Thank you for your enquiry.

My colleagues have just uploaded the SDS document so your customer could get access to the requested form by clinking on the hyperlink.

If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for contacting us.

We have several products from the CytoPainter range which may be of interest to your customer. These can be accessed from the following link:

CytoPainter staining kits | Abcam

These kits allow for the effectively visualization of cellular components and cellular tracking. If the customer is particularly interested in staining both live and dead cells, I would suggest the F-actin CytoPainter kit may be the most suitable. This selectively binds to F-actin and is suitable for fixed tissue sections or cell culture. The kit is available in 4 colours:

https://www.abcam.com/CytoPainter-F-actin-Staining-Kit-Blue-Fluorescence-ab112124.html: Blue (Ex/Em nm 350/450)

https://www.abcam.com/CytoPainter-F-actin-Staining-Kit-Green-Fluorescence-ab112125.html: Green (Ex/Em nm 500/520)

https://www.abcam.com/CytoPainter-F-actin-Staining-Kit-Orange-Fluorescence-ab112126.html: Orange (Ex/Em nm 550/575)

https://www.abcam.com/CytoPainter-F-actin-Staining-Kit-Red-Fluorescence-ab112127.html Red (Ex/Em nm 594/610)

We also have a number of apoptosis assay kits which may be of interest to the customer which can be accessed from the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=13579

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

Thank you for your enquiry.

We recommend Draq5TM, ab108410, for your application, rather than Draq7TM. The laboratory has provided the following notes which describe the differences between the two. In brief, the max emission wavelengths are the same, about 665nm, compared to Tomato Red at about 585nm and propidium iodide at 617nm. It appears that there is some overlap between the DRAQ and Tomato Red emission spectra, but it should be possible to gate out the overlap in the cytometer software.

Click here (or use the following: https://www.abcam.com/DRAQ5trade-ab108410.html).

From the lab:

"We would still recommend the use of DRAQ5 rather than DRAQ7, although you are almost certainly right in your assumption of these nuclei being permeant to DRAQ7 (as they are to PI). However, the issue will be the resolution that can be achieved in the DNA profiles, since this is what you aim to gain by preparing nuclei (Vindelov method) rather than using intact cells, though the separation required here is much less than that for hypoploidy, for example. The likelihood is that DRAQ5 will give better CV's than DRAQ7 and, perhaps, more importantly has a pedigree for DNA content analysis of many papers in the last 10+ years. DRAQ5 can be used to measure DNA content in intact, unfixed cells in complex mixtures such as bone marrow, blood and so will work independent of the permeability of the membranes and as such allows a panoply of control experiments. In spectral terms, DRAQ5 and DRAQ7 are identical."

Please let me know if you have any questions. I will be out next week but one of my colleagues will be able to help.

Thank you for your patience. I have now received the answer in regards to the use of DRAQ7 on yeast. Unfortunately the laboratory does not know neither whether it will work on yeast cells, due to the difference in the cell wall. I am very sorry therefore that we do not know about DRAQ7 suitability for staining of yeast. The laboratory's answer was: “At this time we have no direct experience with DRAQ7 for the detection of membrane-compromised yeast cells. However, I would assume it would work for the detection of such dead and damaged cells but the cell wall is a complication. - It would be possible to use it for fixed yeast cells and also to test for plasma membrane integrity of spheroplasts.” I am sorry for not being more helpful. Please do let me know if you have any questions or concerns.

Thank you for contacting us. Indeed, DRAQ5 has already been used successfully in yeast already. Please see here below the publication citing it: DRAQ5 - http://aem.asm.org/content/72/10/6725.abstract I expect DRAQ7 to be suitable as well for yeast, as it is suitable for most cell types, eukaryotic and prokaryotic. However I have not found an example or publication and have therefore contacted the laboratory to double check. As soon as I get an answer I will forward it to you. 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 inquiry. DRAQ7 is a LIVE cell impermeant far-red DNA dye. It can be used to identify permeable cells in a mix of live and dead cells (i.e. the dead cells) – but in a preparation of fixed cells, all of the cells will be permeant and therefore all will be stained with DRAQ7. If you are trying to differentiate between live and dead cells in a sample (before fixation), then you could simply add DRAQ7 prior to fixation and identify (count) the number of DRAQ7 labelled cells – they will be the dead cells. After fixation, the DRAQ7 will enter all of the cells – and you can now get a total count. Subtraction of dead cell count from total count will give the live cell count.  There are of course issues here – especially if the same sample cannot be used prior to and after fixation. Another idea might be to use DRAQ7 and DRAQ5 (ab108410) in sequence prior to fixation. Adding DRAQ7 first will give the dead cell count. Adding DRAQ5 will then give the total count – and usefully both dyes are in the far-red (same emission). Subtraction of dead count from total count will give the live count. The sample could then be fixed. I hope that this is helpful. Please let me know if you have any other questions.

Thank you for contacting us. I have received the following response from the lab: We would suggest to approach this as follows ... Treat the cell culture with 1-3 uM DRAQ7 during the incubation, image to enumerate the dead/leaky cells at the required end-point and then immediately add 3-5 uM DRAQ5, allow to stain for 5 -10 min and measure again for ALL events, IN THE SAME CHANNEL. The two images will show: 1) dead/apoptotic cells, DRAQ7+ 2) all nucleated cells, DRAQ5+ The two images can be artificially colored to reveal the live cells, when merged. The DRAQ7 positive events can be monitored by imaging in real time during the culture period to observe the progression and dynamics of cell damage and death. Red excitation will limit the risk of DNA damage that is common to UV excited probes. The Draq5 would need being added at the very end of the 24 hour time point, after the last DRAQ7 read. It is tricky to measure the live cells at the beginning of the experiment (i.e. around 0 or 1 hour). We would consider using phase contrast to estimate total cells and subtract the initial DRAQ7 positives. There is no real way to remove Draq5 after incubation for 10 min or so. Once it’s labelled to DNA then it’s there “irreversibly” due to high affinity. As Draq7 is not cytotoxic, an incubation with Draq7 for 24 hours would be fine. DRAQ7 only enters cells that are membrane-compromised. Our data show effectively no toxicity after 96 hours of proximity to proliferative cells. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.