To detect proliferating cells, an alternative thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) was used [24 (link)]. EdU has an alkyne group which enables covalent binding with fluorescent azide via copper-catalyzed reaction, namely, click chemistry. For EdU incorporation, cells were incubated in fresh growth medium supplemented with 15 µM EdU (Lumiprobe, Hannover, Germany; cat# 10540) for 1 h at 37 °C and 5% CO2. Cells were harvested, fixed in 70% ethanol or 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100 in PBS. To label and detect incorporated EdU, cells were incubated with reaction cocktail containing 3 µM sulfo-Cy5-azide (Lumiprobe; cat# A3330), 2 mM CuSO4 (Sigma; cat# 209198) and 20 mg/mL ascorbic acid (Sigma; cat# A4544) in 100 mM Tris-PBS (pH 7.6) for 30 min at room temperature. After washing out the staining cocktail, cell nuclei were stained using 1.5 µg/mL DAPI in 0.1% Triton X-100 in PBS for 30 min at room temperature. For flow cytometry, CytoFLEX (Beckman Coulter) was employed with the setting as follows: 20,000 events for record and 10,000 events for display. DNA replication was analyzed using FlowJo based on histograms of EdU+/− cells. Cell cycle distributions by DNA contents were analyzed using FlowJo with the Dean-Jett Fox algorithm.
5 ethynyl 2 deoxyuridine (edu)
Manufactured by Lumiprobe
Sourced in Germany
EdU is a nucleoside analog that can be incorporated into newly synthesized DNA during cell division. It provides a means of labeling and detecting proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield with dapi, by Vector Laboratories (2 mentions)
Sulfo cyanine5 azide, by Lumiprobe (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
P 97 flaming brown micropipette puller, by Sutter Instruments (1 mentions)
Cy3 azide, by Lumiprobe (1 mentions)
Cyanine 5 azide, by Lumiprobe (1 mentions)
Ro 3306, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Ru486, by Tokyo Chemical Industry (1 mentions)
Incarta software, by GE Healthcare (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Click it plus edu alexa fluor 647 flow cytometry kit, by Thermo Fisher Scientific (1 mentions)
Epifluorescence microscope, by Zeiss (1 mentions)
D9542, by Merck Group (1 mentions)
Cm3050, by Leica (1 mentions)
Camptothecin, by Thermo Fisher Scientific (1 mentions)
Sulfo cy5 azide, by Lumiprobe (1 mentions)
13 protocols using 5 ethynyl 2 deoxyuridine (edu)
1
Detecting Proliferating Cells with EdU
2
Exosome Injection and Cell Proliferation
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A litter of P4 mice was divided into 2 groups, and 2 µL of 2,400×-enriched exosomes (∼3 ± 0.75 µg protein) from DIV9 E18 rat neural cultures were injected into the lateral ventricle (LV) of each cerebral hemisphere. One group was injected with proteinase K-treated exosomes, whereas the other group was injected with control exosomes. Injections were carried out manually with 1.0-mm glass needles (World Precision Instruments) pulled with P-97 flaming/brown micropipette puller (Sutter Instruments). A glass needle with graduated 1-µL volume marks was attached to the syringe with a tube. Intracerebroventricular injections were followed by peritoneal injections of EdU (5-ethynyl-2′-deoxyuridine; Lumiprobe) at a dose of 25 mg/kg by using a Hamilton syringe.
3
Visualizing DNA Synthesis in HeLa Cells
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HeLa cells are cultured according to standard procedures and seeded 1 or 2 days before the treatment on 12 mm diameter #1 glass coverslips. HeLa cells are incubated with 10 μM EdU (5-Ethynyl-2′-deoxyuridine, Lumiprobe) for 30 minutes at 37°C. The cells are fixed with 4% formaldehyde in PBS and permeabilised with 0.1% Triton X-100 in PBS. Click chemistry is performed with 9 μM Cy3-azide (Lumiprobe) and 2 mM CuSO4. To start the reaction, 20 mg/ml ascorbate (final concentration) is added, and the solution is used immediately to stain the cells. After 30 minutes, the cells are washed 3× with PBS, and the sample is incubated with 0.1 μg/ml DAPI for 5 minutes. Samples are mounted in Mowiol (24% glycerol (w/v), 0.1 g mL–1 polyvinylalchol 4.88, 0.1 M Tris-HCl (pH 8.5) in H2O) and used for observation with fluorescence microscopy.
4
Mitosis Detection in Mouse ESCs
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For MiDAS assessment, mESCs were treated with IAA 16–18 hr after passaging. After 18 hr of IAA treatment, mESCs were incubated with 8 µM RO-3306 (Sigma) for 6 hr, washed twice in PBS, and cultured in the presence of 10 µM STLC (Tocris) and 10 µM ethynyl-deoxyuridine (EdU) (Sigma) for 1 hr. mESCs were washed and collected. Chromosome spread preparation was performed as described previously (Pryzhkova and Jordan, 2016 (link)). For EdU detection, chromosome spreads were washed three times in PBS and incubated with ‘click’ reaction cocktail containing 0.1 M Tris (pH 8.5), 10 µM cyanine 5-azide (Lumiprobe), 1 mM CuSO4, and 0.1 M L-ascorbic acid (Sigma) added last. All reaction components were dissolved in 20% dimethylsulfoxide (DMSO) (Sigma) in PBS. Chromosome spreads were incubated with the reaction cocktail for 20 min and washed in PBS with 0.5% Triton three times for 10 min each. Immunocytochemistry was performed as described previously (Pryzhkova et al., 2014 (link)). Antibodies used are listed in Supplementary file 2 . Samples were mounted using Vectashield with DAPI (Vector Laboratories).
5
Genetic Manipulation of Mouse Skin
6
Quantifying Cell Cycle Dynamics by EdU Imaging
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To analyze the percentage of cells in S phase an EdU imaging assay was performed. Briefly, semi-confluent cells were grown in black 96-well plates in the presence of 10 µM EdU (Thermo Fisher Scientific, C10635) for 60 min. After fixation in 4% w/v formaldehyde, permeabilization with 0.2% Triton X-100, EdU labeling with 4 mM Sulfo-Cyanine5 azide (Lumiprobe, A3330) using click chemistry and counterstaining with 5 µg/mL of Hoechst33342, fluorescence intensities for Cyanine5 and Hoechst in single nuclei was measured using an IN Cell 6500 and IN Carta software (GE Life Sciences, GE Healthcare, Chicago, IL, USA) and the ratio of EdU-positive to total nuclei per well was determined with R software.
7
Cell Proliferation Assessment via EdU and Ki-67
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Mice were injected intraperitoneally with 1 mg EdU (Lumiprobe) 2 h before tissue collection for assessment of cell proliferation. EdU detection was carried out using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit (Thermo Fisher Scientific) after surface staining and fixation and permeabilization. Intranuclear staining of Ki-67 was performed in parallel to EdU detection. Cells were analyzed using flow cytometry as described above.
8
EdU Incorporation and p-Rb Staining
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HeLa K cells were plated on 11 mm glass coverslips (Menzel‐Glaser) in 24‐well tissue culture plates. At the end of the experiments, cells were incubated with 10 μM EdU (Lumiprobe, 20540) for 30 min at 37°C, washed once with PBS and fixed for 10 min with 4% paraformaldehyde (PFA) in PBS at RT. Subsequently, the coverslips were incubated in 100 mM Tris–HCl 100 mM pH 7.5 for 5 min at room temperature (RT) and permeabilized with 0.1% Triton X‐100 and 0.02% SDS in PBS for 5 min at RT. The coverslips were rinsed three times with PBS, and the click reaction was performed in freshly prepared label mix (Sufo‐Cy3‐Azide 8 μM [Lumiprobe, B1330], CuSO4 2 mM, ascorbic acid 20 mg/ml [Sigma, A4544] in PBS) for 30 min at RT. Coverslips were subsequently washed three times for 5 min with PBS and mounted on glass slides using Mowiol (Calbiochem) or with 0.75 μg/μl DAPI and imaged with a 63× objective using Zeiss epifluorescence microscope. When combining EdU incorporation assay with p‐Rb staining, after incubation with EdU the immunostaining was performed as for nucleoporin staining (see Immunofluorescence microscopy and sample preparation section, with the difference that post‐fixation was carried out in 4% PFA instead of 1% PFA) and then the click reaction was performed as described previously.
9
Tracking Neurogenesis in Developing Mouse Brain
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Pregnant mouse dams were injected at E13.5 with 1 mg EdU/10 g body weight, delivered as 10 mg/mL solution of 5-Ethynyl-2′-deoxyuridine (EdU, from Carbosynth) in 0.9% sodium chloride saline. Brain tissue was collected from P0 pups by anesthetizing pups, decapitation, 30 min immersion of the head in 4% PFA, and further processing/cryosectioning as outlined earlier in the protocol. To label EdU, slide-mounted sections were rinsed in PBS thrice (rinsed), permeabilized in PBS + 0.5% Triton X-100, rinsed, incubated for 5 min in CLICK reaction cocktail [4 mM CuSO4 pentahydrate, 5 μM Sulfo-Cyanine 5 Azide (Lumiprobe #A3330), 100 mM sodium ascorbate in Tris-buffered saline], rinsed, dried for 30 min, and mounted/coverslipped using Prolong Gold with DAPI.
10
Measuring Cell Proliferation with EdU Labeling
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For cell proliferation experiments, pregnant females were intraperitoneally injected with EdU (25 mg/kg; Lumiprobe) dissolved in PBS. Embryonic samples were collected 1 hour after EdU injection and fixed overnight at 4°C. EdU-treated samples were imaged using the whole-mount IF protocol with the addition of a click reaction step between rehydration and blocking. Samples were incubated in click reaction solution [ascorbic acid (20 mg/ml), 2 mM cupric sulfate, and 4 μM sulfo-Cy3 azide dye (Lumiprobe) in PBS] for 1 hour rocking at room temperature.
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