"It is easy to use and reliable as an internal control"

PRODUCT:                 DRAQ5

Application Area:        In cell western blot (in neural tube coronal slices)

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           3.3

Comments: The product is ideal as an internal control for immunofluorometric quantification using immunofluorescence (infrared range). Results are reproduced consistently.

Name: Anayansi Molina-Hernández

Organisation: INPer, Mexico  

"Very easy to use. High-quality results"

PRODUCT:                  DRAQ5

Application Area:        Cell staining for flow cytometry

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.7

Comments: Very easy to use. High-quality results

Name: Francesco Bellanti

Organisation: University of Foggia

"Great results and publication!"

PRODUCT:                  DRAQ5

Application Area:        Proliferation of cells

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: Easy to use, nice results!

Name: Stéphane Schurmans

Organisation: University of Liège

"The results were good, but DraQ5 was too expensive, its utilization was limited."

PRODUCT:                  DRAQ5

Application Area:        detect the cell nuclei in confocal fluorescent microscopy

Ease of Use:              ⭐⭐⭐⭐

After Sales Service:    ⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐
Average rating:           3.7

Comments: The product worked well. Our main problem with these stains is that they cannot be used in any microscope, e.g. Draq5 can be used in a confocal laser microscope, but cannot be used in another, simpler microscope, so we have to purchase different stains for different microscopes. In addition, DraQ5 is more expensive than other similar stains.

Name: Andras Hrabak

Organisation: Semmelweis University

.........................................

Manufacturer's response: Thank you for your review of DRAQ5. You are right that few epifluorescence microscopes have far-red detection capabilities. You may wish to explore the use of CyTRAK Orange which is excited optimally at 510 nm (488 ex. compatible) and with peak emission at 615 nm (“rhodamine" channel). We do appreciate your concern about pricing.. sadly we have no legal control over end-user pricing by our channel partners. You might, however, consider purchasing direct from BioStatus for the keenest price, fastest service and our expert technical support.

"Great results!"

PRODUCT:                  DRAQ5

Application Area:        Analyze nucleus in confocal microscopy

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.3

Comments: This product is very simple to use and gives high quality result. It also guarantees reproducibility of experiments. I used it for nucleus staining in confocal microscopy at 1:1000 concentration.

Name: Giulio Bontempi

Organisation: University La Sapienza of Roma

"An easy-to-use DNA dye suitable for tissue clearing of mouse brain samples"

PRODUCT:                  DRAQ5

Application Area:        Fluorescence microscopy counterstain for tissue clearing samples

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.7

Comments: The DraQ5 fluorescent probe is supplied as a water-soluble concentrated solution (0.5mM or 5mM) that can be diluted depending on the desired application. Given its high affinity for double-stranded DNA, DraQ5 can be used as a nuclear counterstain in immunohistochemistry studies upon short incubation periods of time, as an alternative to blue-fluorescent DNA-binding probes. However, the broad excitation and emission peaks must be taken into account if a combination with other fluorophores is desired. In our experience, it is also compatible with aqueous-based tissue clearing techniques, rendering a uniform labelling across entire mouse brain hemispheres (55-days-old) that could be imaged by light-sheet fluorescence microscopy. To achieve this, a simple incubation at 37⁰C/60rpm for 12h with a DraQ5 diluted solution (2.5uM) and a 12-hour washing step at 4⁰C to remove excess were incorporated in the tissue clearing procedure.

Name: Miguel Lopes

Organisation: Center for Neuroscience and Cell Biology (CNC)

"excellent product to safely immobilise motile cells"

PRODUCT:                 CyGEL
Application Area:        Live cell fluorescent imaging of Leishmania promastigotes

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:   ⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           4.7

Comments:                 Without Cygel it would be almost impossible to obtain high-quality microscopy of motile forms of Leishmania parasites. The product is easy to use and gives a reproducible performance. I've used it to image Leishmania promastigotes that were modified to contain fluorescent proteins, in order to gain information on protein function. Cellular morphology remained unchanged by the embedding process and protein localization was not affected. Can highly recommend Cygel.

Name: Nathaniel Jones

Organisation: University of York

"Great result"

PRODUCT:                  DRAQ5

Application Area:        Nuclear staining

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           3.3

Comments: The product was easy to use. We achieved high-quality results. We took excellent photos with the confocal microscope. The results were reproducible

Name: Sara Shayan

Organisation: Pasteur Institute of Iran

"It is ok"

PRODUCT:                  DRAQ5

Application Area:        Cancer imaging

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.3

Comments: The effect is ok, but not very stable

Name: Zi Wang

Organisation: CAS

.........................................

Manufacturer's response: Dear Zi, It would be helpful to understand what the issue was in more detail. If staining is not stable over time that can be the case in live cells, where after several hours a re-distribution can occur in some cell types, mostly towards lipophilic structures in the cytoplasm (Martin, 2005). If the staining is not stable in fixed cells then this may be due to washing post-staining, too low concentration, or not removing surfactants used in prior cell permeabilisation procedures.

"great results"

PRODUCT:  DRAQ7
Application Area:  Analyze ROS on tumor cells

Ease of Use:	⭐⭐⭐⭐⭐ (5.0)
After Sales Service:	⭐⭐⭐⭐⭐ (5.0)
Value for Money:	⭐⭐⭐⭐ (4.0)
Average Rating:	4.7

Comments:  Product is simple and effective. We intend using again.

Name: Rodrigo Oliveira
Organisation: Universidade de São Paulo
Review Date: 17 Jun 2022

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"good results"

PRODUCT:  DRAQ7
Application Area:  Flow cytometry

Ease of Use:	⭐⭐⭐⭐⭐ (5.0)
After Sales Service:	⭐⭐⭐⭐⭐ (5.0)
Value for Money:	⭐⭐⭐⭐ (4.0)
Average Rating:	4.7

Comments:  Easy to use. Reliable results.

Name: Michal Simon
Organisation: Kaplan Medical Center
Review Date: 3 May 2022

---

"it works well, minimal crosstalk with other fluorescent channels"

PRODUCT:  DRAQ7
Application Area:  Nuclear counter stain in deep red

Ease of Use:	⭐⭐⭐⭐⭐ (5.0)
After Sales Service:	⭐⭐⭐⭐⭐ (5.0)
Value for Money:	⭐⭐⭐⭐ (4.0)
Average Rating:	4.7

Comments:  It works well, minimal crosstalk with other fluorescent channels. If you have weak signal, use a higher concentration than recommended.

Name: Joshua Morgan
Organisation: University of California, Riverside
Review Date: 21 March 2022

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"Easy to use. Nice results"

PRODUCT:                  DRAQ5

Application Area:        Detection of nuclei in CUBIC cleared samples for light sheet microscopy

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           4.3

Comments: Easy to use. Nice results

Name: Martin MAjer

Organisation: Charles University

.........................................

Manufacturer's response: Thank you so much for the product review, Martin. We value this feedback! We would like to understand how we can do better at after-sales service as we have no record of a direct enquiry, but understand that there maybe at-arms-length improvements to undertake.

"Excellent nuclear stain. Easy to use. Works well with other common stains"

PRODUCT:                  DRAQ5

Application Area:        Cell Staining (nuclei/DNA)

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: DRAQ5 is simple to use since a washing step is not required. It provides a strong signal and good visualization of cell nuclei when used in confocal laser scanning microscopy (CLSM). Its red fluorescent spectrum makes it a suitable stain to work with in conjunction with other common blue and green stains as signal overlap can be avoided.

Name: Joachim Kjesbu

Organisation: Norwegian University of Science and Technology

"Great product and highly reliable"

PRODUCT:                  DRAQ5

Application Area:        Intravital fluorescence microscopy in immune reaction observations

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: The product is perfect to be used in in vivo applications. It allowed cell nuclei segmentation from intravital fluorescence images. OK both in mammalians and chicks.

Name: Claudio Conci

Organisation: Politecnico di Milano, Dip Chimica Mat e Ing. Chimica G.Natta

"Far red cell nucleus stain"

PRODUCT:                  DRAQ5

Application Area:        Cell colocalization analysis

Ease of Use:              ⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐

Value for Money:        ⭐
Average rating:          2.7

Comments: DRAQ5 is generally easy to use. The spectra are in long wavelength that can be paired with many other fluorophores and thus our choice of nucleus dye for confocal imaging. However, the performance is not very stable, sometime it can be very dim and unspecific staining outside the nucleus can be seen. The product (from Thermo Fisher) is very expensive. It will be good if the price can be more reasonable.

Name: Yuning Hong

Organisation: La Trobe University

.........................................

Manufacturer's response: Dear Yuning, thank you for detailed review of DRAQ5. Without seeing examples of the issues you raise it is difficult to give further technical advice. The occasional cytoplasmic staining you describe is often a symptom of understaining or washout due to residual surfactant left over from a prior washing step. Regarding cost, there are a number of other carefully selected suppliers you can choose from and we can advise on the most competitive of these or alternatively order direct from us, the source!

"great results"

PRODUCT:  DRAQ7
Application Area:  Flow cell sorting

Ease of Use:	⭐⭐⭐⭐⭐ (5.0)
After Sales Service:	⭐⭐⭐⭐⭐ (5.0)
Value for Money:	⭐⭐⭐⭐⭐ (5.0)
Average Rating:	5.0

Comments:  This product works very well for flow cell sorting.

Name: Chaoqun Wang
Organisation: UCSF
Review Date: 4 January 2022

---

"Compared with other nuclear dyes, this is one of the best, at least, in our hands"

PRODUCT:                  DRAQ5

Application Area:        Budding yeast nuclear staining of live cells observations

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.7

Comments: DRAQ5 is very easy to use. You resuspend cells in PBS, stain cells in dark for 1-3 minutes, then you can image cells directly (no rinsing is needed). The product is kind of expensive, but it is worth it. The dye can be excited by either 488 or 639 nm laser, but emits in far red, so it won't interfere with GFP.

Name: Zhaojie Zhang

Organisation: University of Wyoming

"Great results"

PRODUCT:                 DRAQ5

Application Area:        Cells and Tissue imaging

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: We achieved high quality and reproducible data.

Name: Cindrilla Chumduri

Organisation: University of Würzburg

"Excellent reproducible results."

 PRODUCT:                 DRAQ5

Application Area:        Fluorescently paint cell nucleus

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: The DRAQ5 reagent is very easy to use. I have used it together with DAPI for counterstaining the cellular nuclei in various cell types like SHSY-5Y, SKN-SH, U2OS and HeLa. The reagent is robust and the staining is perfectly reproducible. I had used the probe in a variety of application like Fluorescent Microscopy, FISH and combined FISH with ICC. The probe lifetime is commendable even after multiple exposures. You can find images with application of DRAQ5 in my publication here - Chanda, K., Jana, N.R. & Mukhopadhyay, D. Receptor tyrosine kinase ROR1 ameliorates Aβ1–42 induced cytoskeletal instability and is regulated by the miR146a-NEAT1 nexus in Alzheimer’s disease. Sci Rep 11, 19254 (2021). https://doi.org/10.1038/s41598-021-98882-0

Name: Kaushik Chanda

Organisation: The Scripps Research Institute, Florida

"Affordable results."

PRODUCT:                 DRAQ5

Application Area:        Flow cytometry

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: The product has worked very well - giving reproducible results.

Name: Sergio Comincini

Organisation: UNIVERSITY OF PAVIA

"very useful stain to interpret microscopy data"

PRODUCT:               CyTRAK Orange

Application Area:      Visualize nuclei and cell cytoplasm by fluorescent confocal microscopy

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:   ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:            5.0

Comments:                 Easy to use stain, relatively inexpensive, good stain to localize intracellular organelles and nuclei.

Name: Leroy Versteeg

Organisation: Baylor College of Medicine

"Great product!"

PRODUCT:                 DRAQ5

Application Area:        Imaging of engineered tissues

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: This product is easy to use and gives us a nice consistent nuclear signal in our engineered tissue models. Since it emits in far-red, it's great for multiplexing with common fluorophores that emit in green/blue/red.

Name: Aleksandra Fomina

Organisation: University of Toronto

"It is one of the best probes I have ever worked on!"

PRODUCT:                 DRAQ5

Application Area:        Cell counting in high-content compound screening

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: We have run high-content compound screening and used this product for cell counting. It works in every experiment:  the staining is simple, rapid and efficient, the signal is strong, it does not interfere with other probes.

Name: Xiaofeng Jiang

Organisation: Harvard University

"Great results."

PRODUCT:                 DRAQ5

Application Area:        Mark cellular nucleus

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.3

Comments: I acquired high-quality results.

Name: Francesco Bellanti

Organisation: University of Foggia

"A necessary tool for immunocytochemistry."

PRODUCT:                 DRAQ5

Application Area:       Immunocytochemical analysis

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.3

Comments: This product is very easy to use and can stain nuclei with high sensitivity.

Name: N/A

Organisation: N/A, Japan

"nice results"

PRODUCT:                 CyTRAK Orange
Application Area:        2-photon live-imaging

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:   ⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.3

Comments:                 We used CyTRAK Orange to label nuclei of acute brain slices and performed 2-Photon live-imaging. The labeling is really easy and fast to process. This dye is very efficient to label nuclei in thick tissue (300 µm) and did not show any toxic effect.

Name: Christelle Cadilhac

Organisation: Université de Montpellier

BioStatus Response:  Dear Christelle, thank you for this review of CyTRAK Orange! We believe this is the first example of its use in 2-photon imaging and it was impressive to see detection through the full depth of 300 micron brain slices.

"Easy & quick"

PRODUCT:                  DRAQ5

Application Area:        In Cell Western - protein normalization

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           4.3

Comments: Had no issues with the DRAQ5 and it produced reliable and repeatable staining of DNA.

Name: Niels Asmussen

Organisation: VCU

"Great results."

PRODUCT:                 DRAQ5

Application Area:        Cell Biology

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: It’s a very good product for our application. It is very easy to use.

Name: Emanuela Senatore

Organisation: Università Federico II Napoli

.........................................

Manufacturer's response: Thank you for your opinion Emanuela! It’s great to have this positive feedback!

"Blue cells."

PRODUCT:                 DRAQ5

Application Area:        Identification of single nucleated cells

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.3

Comments: Easy to use, very potent, just add to your cells and their nuclei will turn blue. Easy peasy lemon squeezy.

Name: hector tejeda

Organisation: erasmus mc

.........................................

Manufacturer's response: Thanks Héctor, for this entertaining review of DRAQ5! You make a good point too: it is possible to view DRAQ5-positive cells with transmission light microscopy - the nuclei are indeed deep blue (as well as far-red fluorescent)! It’s a convenient way to test for functionality and sample quality (for example prior to FACS) and might even allow for a new breed of assays.

"The product is of high quality and easy to use. I would recommend it to everyone."

PRODUCT:                 DRAQ5

Application Area:        Cell staining

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: The product is reliable and works out great. It gives me consistent staining results with high quality. It stains nuclei easily and produces clear images suitable for publication.

Name: Wen Shi

Organisation: University of Nebraska Medical Center

"Great results."

PRODUCT:                  DRAQ5

Application Area:        Live cell nuclear staining

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: Stable and specific signals for live cell nucleus imaging. Very easy to use.

Name: Zhou Zhou

Organisation: Peking University

"The most effective nuclei stain for live cells in our cell type of interest."

PRODUCT:                 DRAQ5

Application Area:        FACS analysis, IF microscopy

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           4.3

Comments: The product was highly effective in helping us identify the nuclei of both live/dead cells. I will be purchasing this product again and believe it to be highly reliable and effective.

Name: Alecia-Jane Twigger

Organisation: University of Cambridge

"Very good."

PRODUCT:                 DRAQ5

Application Area:        Fluorescence imaging

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.0

Comments: I am very satisfied with the product and I often recommend it to other researchers, especially due to its excitation/emission spectrum. In fact, it is very suitable for those who work with autofluorescent scaffolds/biomaterials. It is easy to use, it is a vital dye and marks the cell nucleus well, although sometimes it provides a slightly dirty staining.

Name: Emanuela Jacchetti

Organisation: POLITECNICO DI MILANO

"It is a very good option for labelling cell nuclei."

PRODUCT:                  DRAQ5

Application Area:        Immunohistochemistry

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐
Average rating:           4.3

Comments: This product is very easy to use and always offer reproducible results.

Name: Laura Frago

Organisation: Universidad Autónoma de Madrid 

"Great results!"

PRODUCT:                  DRAQ7

Application Area:        Detect double-strand DNA in tissue samples

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: In our study, cell-impermeable DNA dye DRAQ7 was injected  to explore potential host DNA release. This reagent helped us to prove our experiment could induce DNA release easily.  We have published our paper!

Name: Yan Cao

Organisation: University of Rhode Island 

"Great product, only a little bit pricy!"

PRODUCT:                  DRAQ5

Application Area:        Analyze endosome position respect to the nucleus in mammalian cells

Ease of Use:              ⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐

Value for Money:        ⭐
Average rating:          2.7

Comments: The DRAQ5 is an easy usable marker to stain nucleus. I used it in mammalian cells. It is a really good product, maybe a little expensive.

Name: Giulia Villari

Organisation: university of Turin

.........................................

Manufacturer's response: Dear Giulia, thank you for your valued feedback and independent review. If you have a concern over local pricing we can assist by supplying direct. Also, the small cost of a proven, reliable nuclear counterstain that can be used for 1000-2000 slides should be seen against the significant cost of multiple fluorescently-tagged monoclonal antibodies used alongside it and that its far-red emission enables.

"Our staff prefer DRAQ7 for detecting dead cells using flow cytometry."

PRODUCT:                  DRAQ7

Application Area:        Flow cytometry

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: We have switched to using DRAQ7 for all of our flow cytometry experiments for the detection of dead cells.

Name: Kate Appleton

Organisation: KIYATEC, Inc. 

"Great results."

PRODUCT:                  DRAQ5

Application Area:        Cell nuclei staining

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐
Average rating:           4.7

Comments: Good cell staining to characterize the model by means of confocal microscopy.

Name: Valentina O.

Organisation: Italian Institute of Technology

"Great results."

PRODUCT:                  DRAQ5

Application Area:        Characterization of cell therapy

Ease of Use:              ⭐⭐⭐⭐⭐
After Sales Service:    ⭐⭐⭐⭐⭐

Value for Money:        ⭐⭐⭐⭐⭐
Average rating:           5.0

Comments: Easy to use. Reproducible. Slight improvement would be to have an already diluted form.

Name: Pauline François

Organisation: Assitance Publique Hôpitaux de Marseille AP-HM

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Manufacturer's response: Dear Pauline, thank you for the generous review and the constructive suggestion. We have produced a diluted presentation of DRAQ5 (at 500 µM rather than 5 mM). The product code is DR05500 and, having reviewed your protocol, would perhaps allow you to skip the pre-dilution step entirely e.g. 4 µl into a final volume of 100 µl would give a final concentration of 20 µM. Perhaps this might assist you better?