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DRAQ5™ (ab108410)

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Reviews (5)Q&A (32)References (53)

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Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)
  • Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)
  • Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)

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関連製品

製品の概要

  • 製品名

    DRAQ5™
  • アプリケーション

    適用あり: FM, Flow Cyt, ICC/IFmore details
  • 特記事項

    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

製品の特性

  • 製品の状態

    Liquid
  • 保存方法

    Store at +4°C. Do Not Freeze. Store In the Dark.
  • Concentration information loading...
  • 研究分野

    • Epigenetics and Nuclear Signaling
    • DNA / RNA
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    • Nuclear

アプリケーション

The Abpromise guarantee

Abpromise保証は、 次のテスト済みアプリケーションにおけるab108410の使用に適用されます

アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。

アプリケーション Abreviews 特記事項
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.
特記事項
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.

画像

  • Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)
    Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)

    HeLa cells were stained with Lamin B1 antibody - Nuclear Envelope Marker (ab16048) and alpha Tubulin antibody [DM1A] - Loading Control (ab7291). The cells were 100% methanol fixed (5 min) and then incubated in 1% BSA in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the primary antibodies (ab16048 & ab7291) at 1µg/ml overnight at 4C. The secondary antibodies were Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (DyLight® 488), pre-adsorbed (ab96899) (green) and Goat polyclonal Secondary Antibody to Mouse IgG - H&L (DyLight® 594), pre-adsorbed (ab96881) (red) used at 1/250 dilution for 1h at room temperature. 5µM DRAQ5 was added to the secondary antibody mixture to label nuclear DNA (pseudocolor purple).

  • Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)
    Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)

    HeLa cells were stained with beta Catenin antibody (ab16051) and alpha Tubulin antibody [DM1A] - Loading Control (ab7291). The cells were 100% methanol fixed (5 min) and then incubated in 1% BSA in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the primary antibodies (ab16051 & ab7921) at 1µg/ml overnight at 4C. The secondary antibodies were Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (DyLight® 488), pre-adsorbed (ab96899) (green) and Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (DyLight® 594), pre-adsorbed (ab96899) (red) used at 1/250 dilution for 1h at room temperature. 5µM DRAQ5 was added to the secondary antibody mixture to label nuclear DNA (pseudocolor orange).

  • Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)
    Immunocytochemistry/ Immunofluorescence - DRAQ5™ (ab108410)
    DRAQ5™-stained nuclei in a adult Drosophila brain.

プロトコール

  • Protocol Booklet

Click here to view the general protocols

データシートおよび資料

  • SDS download

  • Datasheet download

    Download

参考文献 (53)

ab108410 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab108410 は 53 報の論文で使用されています。

  • Yap EL  et al. Bidirectional perisomatic inhibitory plasticity of a Fos neuronal network. Nature 590:115-121 (2021). PubMed: 33299180
  • Zhang YW  et al. Human Plasma In-Cell Western Assays-An In vitro Predictor for In vivo Pharmacology in Oncology Drug Discovery. Curr Protoc 1:e51 (2021). PubMed: 33587334
  • SenGupta S  et al. Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-ß and CXCR2 Ligands. Front Immunol 12:659996 (2021). PubMed: 33912188
  • Senatore E  et al. The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation. EMBO J 40:e106503 (2021). PubMed: 33934390
  • Martínez AL  et al. Development of a novel in vitro assay to screen for neuroprotective drugs against iatrogenic neurite shortening. PLoS One 16:e0248139 (2021). PubMed: 33690613
View all Publications for this product

レビューと Q&A

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1-10 of 37 Abreviews or Q&A

DRAQ5: Cell cycle analysis with flow cytometry

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
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

投稿 Aug 22 2018

DRAQ5 In cell Western As Part of an Ab Trial scheme

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
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

投稿 Jun 05 2018

So good!

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

Ms. Sehee Oh

Verified customer

投稿 Jan 31 2018

DRAQ5 co-staining

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

Abcam user community

Verified customer

投稿 Dec 08 2014

DRAQ5 Abcam for In Cell Western Assay

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
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

投稿 Jun 25 2014

Question

I would like to do 2-3 hour live cell imaging with DRAQ5 in murine cells. How long I could image the DRAQ5 stained cells before the cytotoxicity kicks in and cells dye?
Thanks.
Best Regards,

Read More

Abcam community

Verified customer

Asked on Apr 15 2013

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.

Read More

Abcam Scientific Support

Answered on Apr 15 2013

Question

Thank you for your suggestions. I forwarded these to the customer and please see his response:
"I tried the stain on lymphocytes (L1210)- that grew exponentially. and the cycle was very nice - as seen with PI. trying on fixed cells won't help me - I need to get a nice staining on live cells.
the product is kept at 4C all the time and it has 2.5 years till expired.
The cells weren't washed and were analysed 30-60min after staining. All the experiment was conducted with the slowest flow rate. We used all kind of lasers and all gave the same result.
I wasted few days and money on the stain and the FACS experts but to my understanding the product doesn't do what it should do or there is something else that I and the FACS experts in my Faculty don't understand.
Can you send me results from any published paper that used DRAQ5 for this kind of cell cycle staining?"
I appreciate a lot your advise and assistance.
Thanks in advance for the reply.
Kind Regards,

Read More

Abcam community

Verified customer

Asked on May 04 2012

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.

Read More

Abcam Scientific Support

Answered on May 04 2012

Question

ich habe mit Draq5 die Zellkerne von Epithelkarzinomzellen angefärbt und festgestellt, dass die Fluoreszenz mit der Zeit immer stärker wird. Dieser Effekt ist bei höherer Laserpower und bei Einwirkung von Hitze jeweils noch stärker aufgetreten. Woran könnte dies liegen? Binden mit der Zeit immer mehr Farbstoffe an die DNA des Zellkerns? Und fluoresziert er nur, wenn er mit der nuklearen DNA reagiert hat?


Außerdem wollte ich fragen, ob Sie wissen, wie sich der Farbstoff bei der Apoptose verhält. Kann der Farbstoff genauso gut an der DNA des Zellkerns binden, wenn dieser kleiner wird?


Danke im Voraus für Ihre Bemühung!

Read More

Abcam community

Verified customer

Asked on Dec 02 2015

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.

Read More

Anja Hoffmann

Abcam Scientific Support

Answered on Dec 02 2015

Question

I am doing a 18hr cell imaging experiment. When I change the DRAQ5 medium after 12 hours to one without DRAQ5, the staining intensity goes down.

1) Do I need to constantly keep DRAQ5 in my medium even though there might be toxicity effects?

2) How long will the staining last if DRAQ5 is removed from the medium?

Read More

Abcam community

Verified customer

Asked on Aug 06 2015

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.

Read More

達偉 鄭

Abcam Scientific Support

Answered on Aug 06 2015

Question


Questions about DRAQ5 (ab108410) for Flow Cytometry use:



1- After staining with DRAQ5, for how long can the nuclear signal be observed on dead and live cells in a sample?



2- Is there any difference in DRAQ5 nuclear signal lost speedy between dead and live cells? I mean, can DRAQ5 signal be lost faster in dead cells when compared to live cells in the sample?



3- Can DRAQ5 alone be used in FC analysis for differentiate dead cells from live cells in a sample?

Read More

Abcam community

Verified customer

Asked on Aug 13 2013

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.

Read More

Heather Allen

Abcam Scientific Support

Answered on Aug 13 2013

1-10 of 37 Abreviews or Q&A

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