Key features and details
- Assay type: Cell-based
- Detection method: Fluorescent
- Platform: Flow cytometer, Fluorescence microscope
- Sample type: Adherent cells, Suspension cells
Product nameEdU Assay / EdU Staining Proliferation Kit (iFluor 647)
See all Cell viability/proliferation kits
Sample typeAdherent cells, Suspension cells
EdU Assay / EdU Staining Proliferation Kit (iFluor 647) ab222421 provides a sensitive and robust method to detect and quantify cell proliferation in live mammalian cells using flow cytometry or fluorescence microscopy. The iFluor 647 dye (Ex/Em: 649/664 nm) has spectral properties almost identical to those of Cy5®and alternative red fluorophores.
EdU staining protocol summary (wash cells between each step):
- add EdU solution to cells to be stained
- incubate cells for 2-4 hrs under optimal growth conditions
- add fixative solution and incubate for 15 min
- add permeabilization buffer and incubate for 15/20 min
- add reaction mix to fluorescently label EdU and incubate for 30 min
- analyze with flow cytometer / fluorescence microscope
EdU staining can also be combined with antibody staining or cell staining with other fluorescent dyes.
This kit provides enough reagents to perform 50 flow cytometry tests or 50 microscopy tests (for 18 x 18 mm coverslips) or 200 microscopy tests (adapted for 96-well plate format).
Previously called EdU Proliferation Assay Kit (iFluor 647).
The most accurate method to measure DNA proliferation is by directly measuring DNA synthesis. The most common method for this uses antibody-based detection of the nucleoside analog bromo-deoxyuridine (BrdU).
EdU (5-ethynyl-2’-deoxyuridine), a thymidine analog that is an alternative to BrdU, is also used in DNA proliferation assays that are simpler and faster than the BrdU assay. NB: EdU is also available as free molecule as ab146186 (EdU).
In EdU staining, EdU is incorporated into newly synthesized DNA by cells within a sample. A fluorescent azide, such as iFluor-488, is then added. The fluorescent azide is small enough to diffuse freely through native tissues and DNA, and it covalently cross-links to the EdU in a 'click' chemistry reaction.
The main advantages of EdU staining over using BrdU are:
- no harsh DNA hydrolysis / DNA denaturing step is required with EdU staining (unlike in the BrdU assay where it is used to give the BrdU antibody access to BrdU within the DNA)
- EdU staining is faster, and has less steps, than BrdU staining
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
PlatformFlow cytometer, Fluorescence microscope
Storage instructionsStore at -20°C. Please refer to protocols.
Components 50 tests 50 tests 10X Permeabilization Buffer 1 x 25ml 1 x 25ml Azide iFluor 647 Dye (500 µM) 1 x 130µl 1 x 130µl Copper Sulfate (100 mM) 1 x 1ml 1 x 1ml Dimethylsulfoxide (DMSO) 1 x 4.25ml 1 x 4.25ml EdU 1 x 10mg 1 x 10mg Fixative (40% formaldehyde solution) 1 x 5ml 1 x 5ml Sodium Ascorbate 1 x 400mg 1 x 400mg
RelevanceCell proliferation is the multiplication or reproduction of cells, as a result of cell growth and cell division, resulting in the expansion of a cell population.
- Cell Cytotoxicity
- cell tracking
Dot plot of EdU-647 staining (Y-axis, 647) vs FSC. 106 HeLa cells were incubated with the stated concentrations of EdU for 3 hours. Control cells (next image) were incubated with media only. Images were acquired on an Accuri C6 Cytometer (BD Biosciences) with cells excited using a 640 nm laser and data analyzed using FlowJo (v10). The percentage of gated cells (EdU positive) is highlighted.
Dot plot of EdU-647 staining (Y-axis, 647) vs FSC. 106 HeLa cells were incubated with the stated concentrations of EdU for 3 hours. This image shows control cells, incubated with media only. Images were acquired on an Accuri C6 Cytometer (BD Biosciences) with cells excited using a 640 nm laser and data analyzed using FlowJo (v10). The percentage of gated cells (EdU positive) is highlighted.
EdU staining of proliferating cells. HeLa cells (4 x 104 cells/well in 96 plate) were incubated with 20 μM EdU for 3 hours. Cells were analyzed using a TCS SP8 confocal microscope (Leica-Microsystems). DNA (blue) was staining with Hoechst 33342 (ab145597). Purple cells show EdU/Hoechst-positive cells.
EdU staining of proliferating cells. HeLa cells (4 x 104 cells/well in 96 plate) were incubated with 10 μM EdU for 3 hours. Cells were analyzed using a TCS SP8 confocal microscope (Leica-Microsystems). DNA (blue) was staining with Hoechst 33342 (ab145597). Purple cells show EdU/Hoechst-positive cells.
ab222421 has been referenced in 9 publications.
- Wu J et al. NCAPG2 facilitates glioblastoma cells' malignancy and xenograft tumor growth via HBO1 activation by phosphorylation. Cell Tissue Res 383:693-706 (2021). PubMed: 32897418
- Ma X & Liu L Knockdown of FAM225B inhibits the progression of the hypertrophic scar following glaucoma surgery by inhibiting autophagy. Mol Med Rep 23:N/A (2021). PubMed: 33495826
- Wang X et al. CircRNA hsa_circ_0110102 inhibited macrophage activation and hepatocellular carcinoma progression via miR-580-5p/PPARa/CCL2 pathway. Aging (Albany NY) 13:11969-11987 (2021). PubMed: 33891564
- Liang G et al. Overexpression of hsa_circ_0001445 reverses oxLDL-induced inhibition of HUVEC proliferation via SRSF1. Mol Med Rep 24:N/A (2021). PubMed: 33982782
- Gao C et al. MCTS1 promotes the development of lung adenocarcinoma by regulating E2F1 expression. Oncol Lett 22:531 (2021). PubMed: 34079590
- Wang Y et al. Circular RNA circ-SLC7A6 acts as a tumor suppressor in non-small cell lung cancer through abundantly sponging miR-21. Cell Cycle 19:2235-2246 (2020). PubMed: 32794418
- Ding P et al. lncRNA KCNQ1OT1 promotes proliferation and invasion of glioma cells by targeting the miR-375/YAP pathway. Int J Mol Med 46:1983-1992 (2020). PubMed: 33125099
- Xie T et al. microRNA-582 Potentiates Liver and Lung Metastasis of Gastric Carcinoma Cells Through the FOXO3-Mediated PI3K/Akt/Snail Pathway. Cancer Manag Res 12:5201-5212 (2020). PubMed: 32636681
- Khasawneh RR et al. Cross talk between 26S proteasome and mitochondria in human mesenchymal stem cells' ability to survive under hypoxia stress. J Physiol Sci 69:1005-1017 (2019). PubMed: 31679117