EdU is a 5-ethynyl-2′-deoxyuridine analog that is absorbed into dividing cells during DNA synthesis. As a result, EdU inclusion is a marker for cell proliferation. As suggested by the manufacturer EdU Staining Proliferation Kit (iFluor 488) (Abcam, Cambridge, MA, United Kingdom; ab219801). HCT116 and HT-29 cells were seeded in 4 wells (Millicell® EZ Slide, 4-well), and after 72h, cells were treated with a culture medium containing 20 μM EdU reagent. Next, cells were incubated for 2 h and were fixed with paraformaldehyde. Nuclei were stained with DAPI. All image intensities were processed using Image J software through calculating the fluorescence intensity at the DAPI - and GFP-channels and taking their ratio as a quantified read-out for EdU-positivity.
Edu staining proliferation kit ifluor 488
Manufactured by Abcam
Sourced in United Kingdom
The EdU Staining Proliferation Kit (iFluor 488) is a tool for detecting and quantifying cell proliferation. It utilizes the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) to label proliferating cells, which are then detected using a fluorescent iFluor 488 dye. The kit provides the necessary reagents for this assay.
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Lab products found in correlation
Eht1864, by Bio-Techne (1 mentions)
Basic fibroblast growth factor (bfgf), by Miltenyi Biotec (1 mentions)
Pdgf bb, by Miltenyi Biotec (1 mentions)
Il 13, by Miltenyi Biotec (1 mentions)
Il 17, by Miltenyi Biotec (1 mentions)
Tcs sp8 confocal microscope, by Leica camera (1 mentions)
Apollo 567, by RiboBio (1 mentions)
Accuri c6, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
Fcs express 6 plus, by De Novo Software (1 mentions)
Facs canto, by Beckman Coulter (1 mentions)
6 protocols using edu staining proliferation kit ifluor 488
1
EdU Proliferation Assay in Cancer Cells
2
Proliferative signaling in human airway smooth muscle cells
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Primary aSMCs were isolated from human bronchial biopsies. Additional detail on the method is provided in an online data supplement . Human aSMCs were seeded into 24-well plates (10 000 aSMCs/well) and allowed to adhere during 6 hour before serum starvation during 24 hours. When indicated, human aSMCs were treated with the Rac inhibitor, EHT1864 (10−5 M; Tocris Bioscience), P21-activated kinases (Pak) inhibitor IPA3 (10−5 M; Tocris Bioscience), Akt inhibitor VIII (10−5 M; Calbiochem), MEK inhibitor PD98059 (10−5 M; ThermoFisher), JAK inhibitor ruxolitinib (10−5 M; InvivoGen) added 30 min before stimulation with bFGF (25 ng/mL; Miltenyi Biotech), PDGF-bb (25 ng/mL; Miltenyi Biotech), interleukins (IL)-13 (10 ng/mL; Miltenyi Biotech), IL-33 (10 ng/mL; Miltenyi Biotech), IL-17 (20 ng/mL; Miltenyi Biotech), IL-9 (10 ng/mL; Miltenyi Biotech) or TSLP (10 ng/mL; Miltenyi Biotech) for 48 hours. Cells were stained with EdU for 12 hours at 10−5 M according to the manufacturer’s indications (EdU Staining Proliferation Kit iFluor 488, ID: ab219801, Abcam). Proliferation was quantified by the ratio of EdU-positive cells over the total number of cells. Proliferative signalling pathways were analysed by immunoblotting detailed in an online data supplement .
3
EdU Proliferation Assay for Colorectal Cancer
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EdU assays were conducted to assess the viability of CC cells. LOVO and SW620 cells in good growth condition were seeded in a 24-well plate. The culture medium was added with the reagent from an EdU Staining Proliferation Kit (iFluor 488, Abcam Inc., Cambridge, UK) to a final concentration of 10 µmol/L, and incubated in the incubator for 2 h. The cells were then fixed with phosphate buffered saline (PBS) solution containing 4% paraformaldehyde at room temperature for 15 min and incubated with PBS containing 0.5% Triton-100 at room temperature for 20 min. Afterwards, the cells in each well were incubated for 30 min with 100 µL Apollo 567 (Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China) void of light and stained for 5 min with 4ʹ,6-diamidino-2-phenylindole. Five visual fields were randomly taken under a TCS SP8 confocal microscope (Leica, Bannockburn, IL, USA). The blue fluorescence indicates all cells, whereas the red fluorescence is the replicating cells infiltrated by EdU. The rate of EdU-positive cells was calculated.
4
Quantitative Analysis of Cell Apoptosis and Proliferation
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For cell apoptosis assay, after 48 h co-culturing, the cells were collected and washed in ice-cold PBS. PE Annexin V Apoptosis Detection kit (BD Pharmingen™, USA) was used to assess cell apoptosis according to the manufacturer’s instructions. 7-AAD was used to stain dead cells. The cells were incubated with 5 µl of PE Annexin V and 5 µl 7-AAD for 15 min at RT (25 °C) in the dark. Unstained cells, cells stained with PE Annexin V (no 7-AAD) and cells stained with 7-AAD (no PE Annexin V) were used to set up compensation and quadrants.
For the cell proliferation assay, EdU Staining Proliferation Kit (iFluor 488) (Abcam, UK) was used to analyze the proliferation of cells after 48 h co-culturing. EdU solution was added and incubated together with the cells for 4 h under optimal growth conditions. Both cell apoptosis and cell proliferation were analyzed with a flow cytometer (BD Accuri™ C6, BD Biosciences).
For the cell proliferation assay, EdU Staining Proliferation Kit (iFluor 488) (Abcam, UK) was used to analyze the proliferation of cells after 48 h co-culturing. EdU solution was added and incubated together with the cells for 4 h under optimal growth conditions. Both cell apoptosis and cell proliferation were analyzed with a flow cytometer (BD Accuri™ C6, BD Biosciences).
5
Quantifying Cell Proliferation with EdU
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Cell proliferation was determined and quantified by fluorescence microscopy using EdU Staining Proliferation Kit (iFluor 488) (Abcam, Cambridge, MA, USA). Briefly, NPCs were seeded into 96-well plates at a density of 5 × 103 cells/well for 24 h incubation so that they are at 60%–70% confluence. 10 µM EdU solution was prepared to label and culture these cells for 2 h under optimum cell growth conditions. Then, 15 min cell fixation and 20 min permeabilization was performed at room temperature. Subsequently, the reaction mixture of fluorescently labeled EdU was added and incubated for 30 min. EdU positive cells were observed under a fluorescence microscope equipped with an Ex/Em = 491/520 nm filter.
6
Cell Cycle and Apoptosis Assay
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For 5-ethynyl-2′-deoxyuridine (EdU) incorporation, cells were stained according to the manufacturer’s instructions using an EdU Staining Proliferation Kit iFluor 488 (ab219801, Abcam). For cell cycle analysis, the cells were collected using trypsin and suspended in Hanks balanced salt solution buffer with 2% FBS, then washed twice with ice-cold PBS. The cells were then incubated with 100 μg/mL RNase and 100 μg/mL propidium iodide (PI) for 15 min and analyzed using FACS Canto (Beckman Coulter, Brea, CA, USA). Single cells were gated and analyzed to differentiate between G0/G1, S, G2 + M phases, and sub-G0/G1 using FCS Express 6 Plus (De Novo software, Los Angeles, CA, USA). Cell death was measured using annexin V and PI staining. The cells were washed with ice-cold PBS and suspended in 100 μL of staining solution containing 5 μL of annexin V-Alexa-488-conjugate. After 15 min of incubation at room temperature, 400 µL of fresh binding buffer was added per sample containing 1 μL of 20 μg/mL PI, then immediately analyzed using the FACS Canto instrument (Beckman Coulter).
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