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EdU Assay / EdU Staining Proliferation Kit (iFluor 488) (ab219801)

Reviews (1) Submit a question References (25)
EdU Proliferation Kit (iFluor 488) (ab219801)
  • EdU Proliferation Kit (iFluor 488) (ab219801)
  • EdU Prolideration Kit (iFluor 488) (ab219801)
  • EdU Proliferation Kit (iFluor 488) (ab219801)

Key features and details

  • Assay type: Cell-based
  • Detection method: Fluorescent
  • Platform: Flow cytometer, Fluorescence microscope
  • Sample type: Adherent cells, Suspension cells

Overview

  • Product name

    EdU Assay / EdU Staining Proliferation Kit (iFluor 488)

  • Detection method

    Fluorescent

  • Sample type

    Adherent cells, Suspension cells

  • Assay type

    Cell-based

  • Product overview

    EdU Assay / EdU Staining Proliferation Kit (iFluor 488) ab219801 provides a sensitive and robust method to detect and quantify cell proliferation in live mammalian cells using flow cytometry or fluorescence microscopy. The iFluor 488 dye (Ex/Em: 491/520 nm) has spectral properties almost identical to those of FITC and alternative green 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).

  • Notes

    Previously called EdU Proliferation Assay Kit (iFluor 488).

    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.

  • Platform

    Flow cytometer, Fluorescence microscope

Properties

Images

  • EdU Proliferation Kit (iFluor 488) (ab219801)
    EdU Proliferation Kit (iFluor 488) (ab219801)

    Dot plot of EdU-488 staining (Y-axis, 488) vs FSC. 106 HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 488 nm laser and data analyzed using FlowJo (v10). The percentage of gated cells (EdU positive) is highlighted.

  • EdU Proliferation Kit (iFluor 488) (ab219801)
    EdU Proliferation Kit (iFluor 488) (ab219801)

    EdU staining of proliferating cells. HeLa (Human epithelial cell line from cervix adenocarcinoma) 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. Green cells show EdU/Hoechst-positive cells.

  • EdU Prolideration Kit (iFluor 488) (ab219801)
    EdU Prolideration Kit (iFluor 488) (ab219801)

    Dot plot of EdU-488 staining (Y-axis, 488) vs FSC. 106 HeLa (Human epithelial cell line from cervix adenocarcinoma) 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 488 nm laser and data analyzed using FlowJo (v10). The percentage of gated cells (EdU positive) is highlighted.

  • EdU Proliferation Kit (iFluor 488) (ab219801)
    EdU Proliferation Kit (iFluor 488) (ab219801)

    EdU staining of proliferating cells. HeLa (Human epithelial cell line from cervix adenocarcinoma) 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. Green cells show EdU/Hoechst-positive cells.

References (25)

Publishing research using ab219801? Please let us know so that we can cite the reference in this datasheet.

ab219801 has been referenced in 25 publications.

  • Yu YQ  et al. SMYD2 targets RIPK1 and restricts TNF-induced apoptosis and necroptosis to support colon tumor growth. Cell Death Dis 13:52 (2022). PubMed: 35022391
  • Uboveja A  et al. p73-regulated FER1L4 lncRNA sponges the oncogenic potential of miR-1273g-3p and aids in the suppression of colorectal cancer metastasis. iScience 25:103811 (2022). PubMed: 35198876
  • Wik JA  et al. Endogenous glutamine is rate-limiting for anti-CD3 and anti-CD28 induced CD4+ T-cell proliferation and glycolytic activity under hypoxia and normoxia. Biochem J 479:1221-1235 (2022). PubMed: 35695514
  • Yang CH  et al. Independent phenotypic plasticity axes define distinct obesity sub-types. Nat Metab 4:1150-1165 (2022). PubMed: 36097183
  • Li L  et al. LIS1 interacts with CLIP170 to promote tumor growth and metastasis via the Cdc42 signaling pathway in salivary gland adenoid cystic carcinoma. Int J Oncol 61:N/A (2022). PubMed: 36102310
View all Publications for this product

Customer reviews and Q&As

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Ab219801 EdU Proliferation Kit (iFluor 488) in vivo/in ovo

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
Materials Required, Not Supplied
- Double distilled water (ddH2O)
- PBS (phosphate buffered saline)
- 3% BSA (bovine serum albumin) in PBS
- 1X TBS (Tris buffered saline) [50 mM Tris HCl pH 7.5, 150 mM NaCl]
Reagent Preparation
Copper sulfate (100 mM):
- Ready to use as supplied. Equilibrate to room temperature before use. Store at 4°C.
EdU (10 mg):
- Prepare a 10 mM stock solution of EdU by adding 4 mL of PBS to the vial. Mix well by pipetting up and down. Aliquot so that you have enough volume to perform the desired number of assays. Store at -20°C protected from light.
Fixative Stock Solution (40% formaldehyde):
- Make a Working Fixative Solution (4% formaldehyde) by diluting Fixative Stock Solution 1:10 in PBS. Prepare as much as you need for each experiment. Equilibrate to room temperature before use. Store remaining stock solution at 4°C.
iFluor 488 azide dye (500μM in DMSO):
- Ready to use as supplied. Aliquot so that you have enough to perform the desired number of assays. Store at -20°C protected from light.
Sodium ascorbate (lyophilized, 400 mg):
- Make a 10X Stock Solution by dissolving sodium ascorbate in 2 mL of ddH2O. Mix by pipetting up and down until fully dissolved. Label as 10X Additive Solution. Aliquot 10X Stock Solution so that you have enough volume to perform the desired number of assays. Store at -20°C.
10X Permeabilization Buffer (Triton X-100 based):
- Make 1X Permeabilization Buffer by diluting 10X Buffer 1:10 in PBS. Prepare as much as you need for each experiment. Equilibrate to room temperature before use. Store remaining undiluted 10X Buffer solution at 4°C.
Experimental Procedures
Fertilized chicken eggs (HH21 and HH) were incubated at 38 °C in 40-60% humidity atmosphere. The eggs were windowed with scissors, the amnion was disrupted, and the chest was opened. The solution of EdU (300 or 400 µl was pipetted into the open chest). The eggs were taped to prevent dehydration and were reincubated (pulsed) for a further 2 hours. After reincubation, embryos were removed and fixed in 4% PFA/PBS.

1) Prepare 500 µM or 1mM EdU/PBS Solution and 1X Fixative Solution
2) Application of 300 or 400µl EdU (500 µM, 1mM)
3) 2 hod pulsed
4) Fixation – Working Fixative Solution or 4% PFA/PBS 24 hours
5) Paraplast embedding

6) DeWax section – Bioclear/Rotoclear 4x5 min
7) Rehydration 100%, 90%, 80%, 70% EtOH, H2O 2,5 min each
8) Preparation of solutions
a. 1X Fixative Solution (or 4% PFA/PBS)
b. 1x Permeabilization Buffer
c. 3 % BSA/PBS
d. Dilute EdU Additive Solution Reaction Buffer Additive by diluting 10X Additive Solution 1:10 in ddH2O
e. Prepare Reaction mix
9) Permeabilization RT
a. 1x 15 min 4% PFA/PBS
b. Washing 5 min PBS
c. 2x 3 min 3% BSA/PBS
d. Washing 5 min 0,1% Triton
e. 1x 20 min 1X Permeabilization Buffer
f. 2x 3 min 3% BSA/PBS
10) Hybridization RT
a. Prepare Reaction mix for each reaction as described in the table below. 1 slide = 150 µl.
Components Mix for 1 slides (µl) Mix for 4 slides (µl)
1X TBS 0,05M (Tris 50 mM Tris HCl, pH 7,5, 150 nM NaCl) 131,65 525,6
CuSO4 3 12
iFluor 488 azide 0,75 3
Edu additive solution 15 60
Total Volume 150 µl 600 µl
b. Incubation for 30 min, RT, dark.
11) Washing 1x 10 min Permeabilization buffer or PBS
12) Washing 1x 5 min H2O
13) DAPI –1:1000 15 min RT
14) Washing 1x 5 min PBS
15) Washing 1x 5 min H2O
16) Dehydration and mounting in Histokit

Results of Edu labelling of proliferating cells in vivo/ in ovo
To determine the required concentration and volume of EdU solution we used a chicken embryo. We analyzed the staining of proliferating heart cells.
For the experiment, 25 embryos (HH21 and HH31) were used, of which 19 survived. The deaths were always caused by the operator, not by addition of the solution. For the test we used two different concentrations of EdU solutions – 500 µM and 1 mM and two different volumes of EdU solutions – 300 µl and 400 µl. In each case, all samples processed according to the protocol showed positively stained proliferating heart cells. Images of samples from confocal microscope of different concentrations and volume of added EdU solution were recorded with the same parameters and no differences in fluorescence signal were found. Next, coronal section of the head of the chick embryo (HH31) were stained. The EdU signal was present in all parts of the sample, where the Dapi signal was. The labelled parts include the oral cavity, brain, skin, retina and others.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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Submitted Jan 10 2023

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