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An EdU assay measures DNA synthesis by supplying cells with 5-ethynyl-2′-deoxyuridine, a thymidine analogue that is built into new DNA and then detected with a fluorescent azide through a copper-catalysed click reaction. Unlike BrdU detection, it does not need DNA denaturation. The signal marks cells that were in S phase during the labelling window, which is narrower than “proliferating”.
Definition and scope
EdU is “a thymidine analogue in which a terminal alkyne group replaces the methyl group in the 5 position” (Salic and Mitchison, PNAS 2008). Cells that replicate DNA while EdU is present incorporate it in place of thymidine. The alkyne is the handle that the detection chemistry later finds.
In practice “EdU assay” covers any readout built on this label: imaging of fixed cells or tissue, and flow cytometry kits that report the percentage of cells in S phase. Thermo Fisher’s kit documentation frames the purpose as measuring a cell’s ability to proliferate, used for assessing cell health, determining genotoxicity and evaluating anti-cancer drugs, and describes direct measurement of DNA synthesis as the most accurate approach.
How EdU labelling and click detection work
Detection uses a fluorescent azide that reacts with the alkyne of incorporated EdU in a Cu(I)-catalysed [3 + 2] cycloaddition, the reaction family known as click chemistry. The originating paper reports that the reaction can be completed in minutes, with the staining reaction half-maximal in under 5 minutes, and that the small size of the fluorescent azides gives a high degree of specimen penetration compared with antibodies, which allowed whole-mount tissue staining.
Commercial flow cytometry kits use a picolyl azide coupled to dyes such as Alexa Fluor 350, 488, 594 or 647, or Pacific Blue. The manufacturer’s sheet states that standard aldehyde-based fixation and detergent permeabilization are sufficient to give the reagents access to the DNA. So “no denaturation” does not mean “no sample preparation”: the paper’s claim is specifically that, in contrast to BrdU, the method does not require DNA denaturation.
For orientation on neighbouring methods, see our guides on fixing cells and how a flow cytometer works.
EdU vs BrdU vs 3H-thymidine
The kit documentation traces the history: radioactive 3H-thymidine incorporation came first, antibody detection of BrdU replaced it, and EdU kits are presented as alternatives to the BrdU assay.
| Point | EdU | BrdU |
|---|---|---|
| What detects the label | Fluorescent azide, by Cu(I)-catalysed click reaction | Antibody (the method that replaced radioactive 3H-thymidine) |
| DNA denaturation | Not required (Salic and Mitchison 2008); aldehyde fixation and detergent permeabilization are sufficient (Thermo Fisher) | Required: acid, heat, or digestion with DNase (Thermo Fisher) |
| Effect of sample processing | Kit sheet lists compatibility with cell cycle dyes, R-PE and tandems, GFP, RFP and mCherry, and with surface and intracellular antibodies | Processing can alter the cell cycle distribution signal, and acid denaturation can destroy antigen recognition sites (Thermo Fisher) |
Where it matters for labs and kit selection
The manufacturer’s information sheet supports these planning points. They are points to settle before a study, not a bench protocol:
- Titrate for your cells. The kit protocol was developed with Jurkat cells at 10 µM EdU and the sheet says it can be adapted to any cell type, recommending a range of EdU concentrations in initial experiments.
- Concentration and time trade off. The sheet gives a starting point of 10 µM for 1 to 2 hours, with longer incubations using lower concentrations and shorter ones possibly needing higher. Pulse length depends on the growth rate of the cells.
- Include a no-EdU control. The sheet specifies a negative control of the same population not treated with EdU.
- Mind antibody conjugate order. Qdot antibody conjugates should not be used before the click reaction.
- Plan for reagent hazards. The sheet flags DMSO as the carrier, paraformaldehyde as the fixative, and sodium azide in the permeabilization reagent, which can form hydrazoic acid under acid conditions.
- Check the intended use. The kits are labelled “For Research Use Only. Not for use in diagnostic procedures.”
Kit contents in the flow cytometry format are EdU, a dye picolyl azide, DMSO, fixative, a saponin permeabilization reagent, a copper protectant and a buffer additive, stored at 2-8 °C. Panel planning for the cytometry side is covered in our flow cytometry principles and gating guide.
Common misunderstandings
EdU signal is not total proliferation, viability or cell number
The label records DNA synthesis during the labelling window. It does not count cells, and it is not a viability readout. For metabolic readouts see the MTT assay guide and the ATP assay guide; for DNA fragmentation as a different DNA-based endpoint, see the TUNEL assay guide, which also touches on BrdU.
“No fixation” is not the claim
The 2008 paper contrasts EdU with BrdU on fixation and denaturation, while the current kits fix and permeabilize cells with aldehyde and detergent. Read the sentence as “no denaturation”.
It is not a live-cell stain
Salic and Mitchison state that Cu(I) is toxic and the click conditions kill cells, so staining cannot be done in living cells. They also note that chemical modification of DNA could trigger a damage response, so live imaging of labelled cells is of limited utility, and that the staining reaction does not go to completion.
FAQ
What does EdU stand for?
EdU is 5-ethynyl-2′-deoxyuridine, a thymidine analogue with a terminal alkyne group in place of the methyl group at the 5 position.
Does an EdU assay need DNA denaturation?
No. The originating paper states that, in contrast to BrdU, the method does not require DNA denaturation, and the Thermo Fisher sheet says aldehyde fixation with detergent permeabilization is sufficient.
Can EdU be used in live cells?
Not for the click staining step. The paper states that Cu(I) is toxic and the click conditions kill cells.
Can EdU detection be combined with GFP or antibodies?
The Thermo Fisher flow cytometry kit sheet lists compatibility with GFP, RFP, mCherry, cell cycle dyes, R-PE and tandems, and surface and intracellular antibodies, with the caution not to use Qdot antibody conjugates before the click reaction.
Sources
- Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of DNA synthesis in vivo. PNAS 2008;105(7):2415-2420. pmc.ncbi.nlm.nih.gov/articles/PMC2268151 (checked 2026-10-29).
- Thermo Fisher Scientific. Click-iT Plus EdU Flow Cytometry Assay Kits, product information sheet, Pub. No. MAN0009883 Rev. E.0. documents.thermofisher.com (checked 2026-10-29).
