DU145 and PC3 cells (5×105 cells/cell) were seeded into six-well plates at 37°C 24 h before transfection was performed and were incubated overnight to reach 30–50% confluence. Transfection was then performed for 48 h. EdU solution (Guangzhou RiboBio Co., Ltd.) was subsequently diluted using RPMI-1640 medium at a ratio of 1:1,000 to prepare a 50 µM EdU solution. When 80–90% confluency was reached, each well was treated with 100 µl 50 µM EdU solution for 2 h at room temperature, following which 4% paraformaldehyde was added to each well and the cells were incubated for 15–30 min at room temperature. The cells were then incubated with 2 mg/ml glycine for 2 min, then 100 µl detergent (0.5% Triton X-100 in PBS) was used to permeabilize cells in each well at room temperature for 5 min. A total of 100 µl 1X Apollo®567 staining solution (cat. no. C10316-1; Guangzhou RiboBio Co., Ltd.) was added to each well, where the cells were then protected from light and incubated for 30 min at room temperature. Finally, 100 µl 1X Hoechst 33342 reaction solution (Guangzhou RiboBio Co., Ltd.) was added to each well in the dark for 10–30 min followed by observation and analysis using a fluorescent inverted microscope (Magnification, ×100; DMI6000B; Leica Microsystems GmbH. Images were analyzed using the Image J software (version 1.44p; National Institutes of Health).
Edu solution
Manufactured by RiboBio
Sourced in China
EdU solution is a nucleoside analog that can be used to label and track proliferating cells. It is incorporated into DNA during the S phase of the cell cycle, allowing for the identification of actively dividing cells.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Cck 8 solution, by Dojindo Laboratories (2 mentions)
Fluorescence microscope, by Zeiss (2 mentions)
Fluorescent microscope, by Leica (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Triton x 100, by Solarbio (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Annexin 5 phycoerythrin pe apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
7 aad flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Edu apollo in vitro imaging kit, by RiboBio (1 mentions)
Methanol, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bz 8000 fluorescence microscope, by Keyence (1 mentions)
Dapi solution, by RiboBio (1 mentions)
Cck 8 reagent, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Crystal violet, by Merck Group (1 mentions)
47 protocols using edu solution
1
Quantifying Cell Proliferation with EdU Assay
2
Cardiac Fibroblast Proliferation Assay
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Cardiac fibroblasts were seeded in 24-well plates and added with EdU solution (Ribobio, China) on accordance with the protocols. The cells were incubated for 2 h, and then treated and photographed.
3
Cell Proliferation Assay with EdU
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A total of 4×104 cells were cultured in 96-well plates and exposed to 50 µM EdU solution (Guangzhou RiboBio Co., Ltd.) for 4 h at 37°C and then fixed in 4% formaldehyde overnight at 4°C and permeabilized in 0.5% Triton X-100 for 10 min. Cells were incubated with 100 µl Apollo reaction cocktail for 30 min at room temperature in the dark and DNA was stained with Hoechst 33342 (100 µl/well) for 30 min at room temperature in the dark. The stained cells were analyzed using a fluorescent microscope (6 fields were selected randomly; magnification, ×40).
4
Cell Proliferation Assessment by MTT and EdU
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Cell proliferation assays were performed by MTT and EdU analyses. After cell treatment, MTT assay was performed according to the instructions. Cells were incubated in 96-well plates with 200 µL of 5 mg/mL MTT (Sigma, St. Louis, MO, USA). After 4 h, 150 µL of DMSO was added into the plates after the medium was aspirated.
For EdU assay, the treated cells were exposed to 10 nM of EdU solution (RiboBio, Guangzhou, China). After 2 h, cells were then treated by 4% formaldehyde and 0.5% Triton X-100. After 30 min of treatment with Apollo cocktail, Hoechst was applied to stain cells for 30 min. The fluorescence microscopy was used to observe and record the images.
For EdU assay, the treated cells were exposed to 10 nM of EdU solution (RiboBio, Guangzhou, China). After 2 h, cells were then treated by 4% formaldehyde and 0.5% Triton X-100. After 30 min of treatment with Apollo cocktail, Hoechst was applied to stain cells for 30 min. The fluorescence microscopy was used to observe and record the images.
5
Assessing Cited2 Effects on Y-/O-CSC Apoptosis and Proliferation
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Y−/O-CSCs were transducted with Cited2-shRNA, Cited2-cDNA, and negative control lentiviral particles; 48 h later, cell apoptosis was detected by flow cytometry using an Annexin V-phycoerythrin (PE) Apoptosis Detection kit (Invitrogen, Carlsbad, CA, USA). The percentage of apoptotic cells was counted as the sum of annexin V-PE single-positive and annexin V-PE/7-aminoactinomycin D (7-AAD) double-positive cells. The 5-ethynyl-2′-deoxyuridine (EdU) assay was performed to assess cell proliferation 48 h after lentiviral infection by incubating the cells in 10 μmol/l EdU solution (RiboBio, Guangzhou, China) for 2 h followed by flow cytometry analysis. Cells cycle analysis was performed with the 7-AAD Flow Cytometry Assay kit (Ebioscience, San Diego, CA, USA) according to the manufacturer’s instructions. The methods have been described in detail elsewhere [11 (link), 12 (link)].
6
Quantifying Cell Proliferation using EdU
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Lentivirus-transfected GC cells were cultured in 96-well plates. First, 200 µL of a 50 µmol/L EdU solution (Guangzhou RiboBio Co.,LTD, China) was added to the cultured cells. After washing, fixation, and permeabilization, 100 µL of 1× Apollo solution was added to the cells. Cellular DNA was then stained with 100 µL of 1× Hoechst 33342 solution. A fluorescence microscope was used to capture images of EdU- and Hoechst 33342-positive cells.
7
Cell Proliferation Assay using EdU
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Cell proliferation was further determined by EdU Apollo In Vitro Imaging Kit (Ribobio, Guangzhou, China). Briefly, cells were incubated with EDU solution (Ribobio) for 2 h. Supernatant was discarded, and cells were fixed with methanol (Millipore) for 30 min. After that, cells were incubated with glycine, 0.5% TritonX-100 and Hoechst 33342 reaction buffer, respectively. Finally, samples were assessed by Fluorescence microscopy.
8
Cell Proliferation Analysis with EdU and CCK-8
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For the evaluation of cell proliferation, 5-ethynyl-2′-deoxyuridine (Edu) labeling and Cell Counting Kit-8 (CCK-8) assays were performed. Briefly, for Edu assay, transfected cells were incubated with Edu solution (50 μM, Ribobio) for 2 h, and subsequently, the Edu-labeled cells were stained with 1 × Apollo567 (Ribobio) for 30 min. After being staining with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) for nuclei staining, the cells were observed under a BZ-8000 fluorescence microscope (Keyence, Osaka, Japan). The ratio of Edu-positive nuclei (red) to total nuclei (blue) was determined as the proliferation rate of transfected cells in 10 randomly captured fields per well. For CCK-8 assay, ~ 2500 transfected cells were plated into each well of 96-well cell culture dishes and incubated in complete medium at 37 °C. The cells were evaluated for potential growth every 24 h using the CCK-8 solution (10 μL per well) as per the manufacturing guidance (Dojindo, Kumamoto, Japan). After addition of CCK-8 solution, a 2-h incubation was allowed at 37 °C. A 96-well spectrometer (BMG Labtech, Ortenberg, Germany) was used to measure the absorption with a 450 nm filter.
9
Assessing VSMC Proliferation: CCK-8 and EdU
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Cell Counting Kit-8 (CCK-8) assay was performed to assess VSMC proliferation. Cells were incubated in DMEM at a density of 5 × 103 cells per well in 96-well plates for 24 h after transfection. And then the cells were maintained in 10 μl /well CCK-8 solution (7Sea-Cell Counting Kit, Shanghai, China) for an additional 1 h. Finally, the value was measured at 450 nm absorbance.
Another way to test cell proliferation is to use the EdU assay, VSMCs were cultured with EdU solution (50 nmol/L) (RiboBio, Guangzhou, China) for 2 h, then VSMCs were stained according to the product instructions. And finally, the pictures were obtained by a fluorescence microscope (Zeiss, LSM510, META). Image J 1.8.0 was used for analysis of the data.
Another way to test cell proliferation is to use the EdU assay, VSMCs were cultured with EdU solution (50 nmol/L) (RiboBio, Guangzhou, China) for 2 h, then VSMCs were stained according to the product instructions. And finally, the pictures were obtained by a fluorescence microscope (Zeiss, LSM510, META). Image J 1.8.0 was used for analysis of the data.
10
Evaluating Breast Cancer Cell Proliferation
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EdU assay was conducted to further evaluate the effects of PER1 expression on the proliferation of breast cancer cells. In brief, transfected cells were seeded into 24-well plates at a density of 5000 cells/well. After that, cells were incubated in medium with EdU solution (RiboBio, China) for 2 h, and then were fixed with 4% paraformaldehyde and stained by DAPI. The percentage of EdU-positive cells was counted under a fluorescence microscope (Olympus, Japan).
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