- Find MSDS or SDS, a COA, data sheets and more information. The proliferation signal from A375 melanoma cells expressing an Erk2- GFP fusion was detected using either BrdU or Click-iT Plus EdU The ability to multiplex Click-iT Plus EdU assays with other fluorescent probes opens the door to a more complete analysis of cell function. 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. If the ingredients are not added in the order listed, the reaction may not proceed optimally or might fail. Not for use in diagnostic or therapeutic procedures.

For the preparation of 500 mL of 1× saponin-based permeabilization buffer and wash reagent, add 50 mL Component D to 450 mL 1% BSA in 1× PBS. The cell culture density should not exceed 2 × 10^6 cells/mL.Incubate the treated cells for the desired length of time.

In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently lab

We have found a final concentration of 10 μM EdU to be sufficient for labeling of human and mouse cell lines and primary cell populations.During addition of EdU to cells in culture, avoid disturbing the cells in any way (eg, centrifugation steps or temperature changes) that may disrupt the normal cell cycling patterns.

2008; 44(7):927-929. We recommend that all controls, including unstained and single stained controls, also be treated with the click cocktail with or without the dye azide as appropriate so that control and test samples exhibit the same autofluorescent properties. When compared with traditional antibody-based BrdU methods, the Click-iT Plus EdU cell proliferation assays not only offer better performance and an easier workflow but are now compatible with an even broader range of commonly used fluorescent probes, including GFP, RFP, and other fluorescent proteins as well as phycobiliproteins.The Click‑iT Plus EdU assay represents a significant breakthrough in the evolution of cell proliferation measurements Although it eliminates the complications of working with radioactivity, the BrdU proliferation assay is both difficult to perform consistently and time consuming, typically requiring 6–24 hr to complete. We recommend preparing aliquots to avoid repeated freeze-thaw cycles.For Eterneon™ Red 645 azide, we recommend preparing aliquots to avoid repeated freeze-thaw cycles. The cell culture density should not exceed 70% confluence.Incubate the treated cells for the desired length of time.

When co-stained with a DNA dye such as 7-AAD, PI, or DAPI, cell populations may be segmented by flow cytometry into the G0/G1-phases (2N DNA content, EdU-negative), S-phase (2N-4N DNA content, EdU-positive), or G2/M-phases (4N DNA content, EdU-negative).The BD Pharmingen™ 647 EdU Click Proliferation Kit contains Eterneon™ Red 645 Azide, which is excited by the red laser and has an excitation maximum of 643 nm and an emission maximum of 662 nm. Permeabilization Buffer volume should be large enough to cover the well entirely (eg, at least 50 μL for a 96-well).Incubate samples for 15 minutes at room temperature.Make up a working solution of the dye azide (Component B) by diluting 1:5 with DMSO (eg, dilute 1 μL dye azide with 4 μL DMSO). Proc Natl Acad Sci U S A.

Vortex gently to mix.This assay was optimized for 96-well plate imaging.

No. An EdU concentration of 10 µM is a good starting concentration.

Kolb HC, Finn MG, Sharpless KB.

Not for use in diagnostic or therapeutic procedures. 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. We recommend preparing aliquots to avoid repeated freeze-thaw cycles.

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.

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.

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. Click Chemistry: Diverse Chemical Function from a Few Good Reactions.

For Research Use Only. After use, store any remaining solution at 2 - 8°C.

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