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In addition to the imaging and flow cytometry kits, the Click-iT Plus technology is available in the Click-iT Plus Alexa Fluor Picolyl Azide Toolkits, which contain the reagents needed to perform copper-catalyzed click reactions with copper-sensitive compounds. EdU-647 100 assays

If the ingredients are not added in the order listed, the reaction may not proceed optimally or might fail.







Proliferation, however, is rarely assayed in isolation; other cell function probes are often used in concert with proliferation assays to provide a more informative picture of the state of the cell. Click-iT Plus EdU Proliferation Kits for Imaging and Flow Cytometry





EdU Cell Proliferation Assay (EdU-647) The EdU-647 Cell Proliferation Assay is a superior alternative to conventional BrdU assays.

Eterneon™ Red 645 can be read out of filters commonly used for APC or Alexa Fluor® 647 (eg, 670/30 bandpass filter). Plant Methods. Vortex well to mix.Prepare the assay cocktail as described in Table II after section G, adding reagents in the same order indicated in the table. Wang Q, Chan TR, Hilgraf R, Fokin VV, Sharpless KB, Finn MG. Bioconjugation by Copper(I)-Catalyzed Azide-Alkyne [3 + 2] Cycloaddition.

Analyte Specific Reagent. We recommend preparing aliquots to avoid repeated freeze-thaw cycles. Biotechniques. In this method, the thymidine analog EdU (5-ethynyl-2´-deoxyuridine) is incorporated into newly synthesized DNA by cells progressing through the S (DNA synthesis) phase of the cell cycle.

554656) Fluorescent antibodies or dyes for immunophenotypic or functional analysis (optional)Red laser-equipped cytometers (BD FACSCanto™ II, BD LSRFortessa™, BD LSR™ II, and BD Accuri™ C6 Flow Cytometer) can be used. Detection of S-phase cell cycle progression using 5-ethynyl-2’-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2’-deoxyuridine antibodies.

General Purpose Reagent

If DNA content is to be assessed, samples should be acquired on a low flow rate to generate the best resolution of 2N and 4N populations.Allow all vials to warm to room temperature before opening.For the preparation of a 10 mM stock solution of EdU, add 4 mL DMSO or 1× PBS to EdU and mix until completely dissolved.


Component B, Eterneon™ Red 645 azide (10 mM), Material Number 51-9012111, contains 99.4% DMSO.Keep away from flames and hot surfaces.





In this case, dilute an aliquot of Component B with an appropriate amount of DMSO and add 1 μL of the diluted dye azide in place of the concentrated stock.Add 500 μL of the assay cocktail to each sample and vortex gently to mix.Incubate the samples for 30 minutes at room temperature protected from light.Wash samples by adding 2 mL 1× Saponin-Based Permeabilization Buffer (prepared in step C5), pelleting by centrifugation, and removing the supernatant.If you do not wish to stain for intracellular antigens or DNA content, dislodge the cell pellet and resuspend cells in 500 μL 1× PBS or equivalent, and proceed to analysis on the flow cytometer.Staining of Intracellular and/or Surface Antigens (Optional)Dislodge the cell pellet and resuspend cells in 100 μL BD Pharmingen™ Stain Buffer.Add fluorescent antibodies specific for intracellular and/or surface markers to tubes and vortex gently to mix.Because the click reaction cocktail contains copper and copper is known to quench the fluorescence of R-PE, antibody conjugates containing R-PE or R-PE tandems should be stained at this point in the procedure.Incubate samples for 30 minutes at room temperature or on ice.Wash samples by adding 1 mL BD Pharmingen™ Stain Buffer, pelleting by centrifugation, and removing the supernatant.Dislodge the cell pellet and resuspend in 500 μL 1× PBS containing an appropriate amount of DNA dye (eg, DAPI, PI, or 7-AAD) for your cell type of interest. They allow you to optimize your own Click–iT Plus detection of alkyne-containing biomolecules Download a hyperlink-enabled, printer-friendly version of this article.For Research Use Only.

As a starting point, we recommend 10 μM EdU for 1 hour. Once the assay cocktail is prepared, use it within the next 15 minutes.Please note that in some cases, titration of the dye azide (Component B) can increase resolution of EdU positive and negative populations.

Cell type variations, cell density, growth medium and other factors may influence the labelling. Aliquots should be stored dry and protected from light at -20°C.For the preparation of a 10× stock solution of the buffer additive, add 4 mL deionized water to the buffer additive and mix until completely dissolved. 2001; 40(11):2004-2021.

Please note that this kit is provided as Part 1 of 2 (Components A, B, and C) to be stored dry and protected from light at -20°C, and Part 2 of 2 (Components D, E, F, and G) to be stored dry at 2 - 8°C or room temperature.


Kotogany E, Dudits D, Horvath GV, Ayaydin F. A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine.

See "Kit Contents" immediately below for more information.Saponin-based Permeabilization and Wash Reagent (10×)Please note that Hazard Warnings for components listed above are found on Page 5 of this document.Buffered saline solution, such as PBS, DPBS, or TBSBD Pharmingen™ Stain Buffer (BSA) (Cat. Harvest cells, pellet by centrifugation, and remove EdU-containing media.Dislodge the cell pellet, resuspend cells in BD Pharmingen™ Stain Buffer (BSA) (Cat.

Not for use in diagnostic or therapeutic procedures.

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