Human SSCs were seeded at 5000 cells/well in a 96-well plate; each well contained DMEM/F12 medium containing 50 μmol/L EdU (RiboBio). The cells were cultured for 12 h, washed using DMEM, and subjected to fixation using 40 g/L paraformaldehyde. Cell neutralization was achieved using 2 mg/mL glycine, followed by permeabilization for 10 min using 5 mL/L Triton X-100 at room temperature. Apollo staining reaction buffer was then used to reveal the EdU staining. Cell nuclei were stained using Hoechst 33342. A fluorescence microscope (Zeiss) was used to capture images, and the positive rate of EdU staining was calculated by counting at least 500 cells.
5 ethynyl 2 deoxyuridine (edu)
Manufactured by Zeiss
Sourced in Germany
EdU is a thymidine analog that is used as a marker for cell proliferation. It incorporates into DNA during the S-phase of the cell cycle and can be detected using specific antibodies.
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Lab products found in correlation
5 ethynyl 2 deoxyuridine (edu), by RiboBio (4 mentions)
Lsm 880, by Zeiss (4 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (3 mentions)
Inverted fluorescence microscope, by Zeiss (2 mentions)
Apollo dye solution, by RiboBio (2 mentions)
Hoechst, by RiboBio (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Fluorescence microscopy, by Zeiss (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Carl microscope, by Zeiss (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Keygen Biotech (1 mentions)
Click it reaction mixture, by Keygen Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Metafer4, by MetaSystems (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Prism 8, by GraphPad (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Tokyo Chemical Industry (1 mentions)
12 protocols using 5 ethynyl 2 deoxyuridine (edu)
1
Quantifying Human Stem Cell Proliferation
2
EdU Proliferation Assay Protocol
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Cells (5 × 104) were seeded in 24-well plates and cultured overnight. Then 300 µl EdU (50 µM)(RiboBio, China) was added in each well and incubated for 2 h. Then cells were fixed by 4% paraformaldehyde for 20 min and permeabilized by 0.5% TritonX-100 (Beyotime, China) for 20 min, dyed in 300 µl Apollo dye solution (RiboBio, China) for 25 min. Cell nuclei were dyed with Hoechst (RiboBio, China) for 10–30 min. Cells were observed under an inverted fluorescence microscope (Zeiss, Germany) at a magnification of 200×. And then proportion of EdU positive cells was calculated.
3
Quantifying RGC Differentiation in Vitro
4
EdU Incorporation Assay for Glioma Cells
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Cells (75,000) were plated in 24-well plates with round coverslips overnight. 200 µl of EdU (10 µM, KeyGEN Bio TECH, Nanjing, China) were added to each well, and the cells were incubated at 37°C, 2 h. The cells were fixed by adding 4% neutral paraformaldehyde to each well and removed after 30 minutes at room temperature. Then, we added 0.5% TritonX-100 (KenGEN, Nanjing, China) in PBS (200 µl) to each well for 20 min and stained the samples with 200 µl Click-iT reaction mixture (KeyGEN Bio TECH, Nanjing, China) for 30 min under lightproof conditions. Next, DAPI (Beyotime, China) was used to stain the glioma cell nuclei. Round coverslips were observed using Carl Zeiss microscope (Carl Zeiss, Germany) to analyze the proportion of EdU-positive cells.
5
Cell Cycle-Specific DSB Repair Analysis
6
Quantifying Neuronal Proliferation via EdU Labeling
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A thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU, Tokyo Chemical Industry) was intraperitoneally injected into a pregnant mouse (50 mg/kg body weight) at the indicated time points before or after TM injection. The brains were dissected from the mice at P0, and coronal sections of 16-μm thickness were prepared as explained above. The sections were antigen-retrieved as described above and immunostained with chicken anti-βGAL and donkey Alexa488-conjugated anti-chicken IgY antibodies. Subsequently, the incorporated EdU was detected in a solution of 0.1 M Tris (pH 7.6), 2 mM CuSO4, 3 μM Alexa555 azide triethylammonium salt (A20012, Thermo Fisher Scientific), and 10 mM ascorbic acid for 40 min at room temperature. The numbers of neurons labeled for βGAL and those doubly labeled for βGal and EdU were manually counted for each mouse with a 40× objective lens (Plan-Apochromat) under a fluorescent microscope (Zeiss Axioplan2) with 38 HE (ex470/40, em525/50) and 15 (ex549/12, em590) filter sets. The exact numbers of animals and neurons used for quantification are shown in the figures and legends. The scattered plots (Figures 5 C–5F and 7 C–7F) were generated using the Prism8 software (GraphPad).
7
Evaluating DLBCL Cell Proliferation
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First, OCI-Ly3 and OCI-Ly10 cells were treated with 1-L-MT for 48 hours. DLBCL cells were then incubated with 50 μM EdU (RiboBio, C10310-3, Guangzhou, China) for 2 hours at 37 °C. After fixation with 4% paraformaldehyde and washing with PBS, the cells were stained with 1× Apollo for 30 mins in the dark. Then, 1× Hoechst 33342 was used to stain cell nuclei. Finally, the EdU-stained cells were centrifuged and resuspended in glass-bottom cell culture dishes, and images were acquired by a confocal microscope (LSM880, Carl Zeiss, Oberkochen, Germany).
8
Cell Proliferation Assay with EdU
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OCI-Ly3 and OCI-Ly10 cells were seeded into 12-well plates at a density of 5×105 cells/well. Besides, 48 h after stimulation with Apcin, the cells were exposed to 50 μM EdU (C10310-3; RiboBio, Guangzhou, China) for 2 h at 37 ºC. Then, the cells were stained with 1 × Apollo and 1 × Hoechst 33,342 according to the manufacturer’s instructions. Finally, the EdU-stained cells were visualized under a confocal microscope (LSM880; Carl Zeiss, Oberkochen, Germany) with excitation and emission wavelengths of 488 and 520 nm, respectively.
9
Quantifying Cell Proliferation
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Transfected CaSki or SiHa cells were seeded into fresh 96-well plates for cultivation. After incubation with EdU (Sigma-Aldrich, St. Louis, MO, USA) for 2 h, the cells were fixed by using 4% PFA (Solarbio, Beijing, China) and dyed in Apollo Dye Solution (RiboBio, Guangdong, China). Nucleic acid was stained with Hoechst 33342 stain (Invitrogen, Carlsbad, USA). Images were captured under an inverted fluorescence microscope (Carl Zeiss, Jena, Germany) and the proportion of EdU-positive cells was determined.
10
Exosome Uptake and Cell Proliferation
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First, the cells were placed in a 24-well plate, and PC-3 and 22RV1 cells were grown evenly in a well plate.
After the cells adhered to the wall, exosomes labeled with PKH26 were added, and the cell crawl sheets were removed after 24 h for paraformaldehyde xation, followed by staining of the cytoskeleton with phalloidin, labeling of the nucleus with DAPI, and observation under a uorescence microscope (Zeiss, Germany).
EdU cell proliferation assay PC-3 and 22RV1 cells were spread evenly in 24-well plates containing cell crawlers, and when the cell density reached 70-80%, complete medium containing EdU solution (Beyotime, China) was added and incubated for 2 h at 37°C. The EdU medium was removed, wash with PBS three times (5 min each time), xed with 4% paraformaldehyde at room temperature for 15 min, and washed with PBS three times (5 min each time). PBS containing 0.3% TritonX-100 was added, and the cells were incubated at room temperature for 10 min. The permeation solution was removed, and cell were washed with PBS, stained with DAPI, and observed under a uorescence microscope.
After the cells adhered to the wall, exosomes labeled with PKH26 were added, and the cell crawl sheets were removed after 24 h for paraformaldehyde xation, followed by staining of the cytoskeleton with phalloidin, labeling of the nucleus with DAPI, and observation under a uorescence microscope (Zeiss, Germany).
EdU cell proliferation assay PC-3 and 22RV1 cells were spread evenly in 24-well plates containing cell crawlers, and when the cell density reached 70-80%, complete medium containing EdU solution (Beyotime, China) was added and incubated for 2 h at 37°C. The EdU medium was removed, wash with PBS three times (5 min each time), xed with 4% paraformaldehyde at room temperature for 15 min, and washed with PBS three times (5 min each time). PBS containing 0.3% TritonX-100 was added, and the cells were incubated at room temperature for 10 min. The permeation solution was removed, and cell were washed with PBS, stained with DAPI, and observed under a uorescence microscope.
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