A CCK8 kit (Biosharp, CA, USA) was utilized to detect the cell viability. The transfected cells were planted in 96-well plates with 5000 cells per well. After a period of culture, these cells were incubated with CCK8 reagent for 1 h. Finally, the absorbance (450 nm) was measured and analyzed using a microplate reader (Molecular Devices, CA, USA). For the EdU assay, after incubation with the EdU solution (Beyotime, Shanghai, China) in a dark environment at 37 °C for 1 h, the cells were immobilized with paraformaldehyde for 20 min, and DAPI (Sigma, Shanghai, China) was utilized to visualize the nucleus. Subsequently, EDU-positive cells were captured and counted using an Olympus fluorescent microscope.
Edu solution
Manufactured by Beyotime
Sourced in China
EdU (5-ethynyl-2'-deoxyuridine) solution is a compound used in cell biology research. It is a synthetic nucleoside analog that can be incorporated into DNA during DNA synthesis, allowing for the detection and visualization of proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (5 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Cck 8, by Solarbio (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Elx808 microplate reader, by Agilent Technologies (2 mentions)
Cck 8 kit, by Biosharp (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Methanol, by Sangon (1 mentions)
Giemsa, by Beyotime (1 mentions)
Cck 8 reagent, by Beyotime (1 mentions)
Triton x 100, by Sangon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Yeasen (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Triton x 100, by Dow (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Edu solution, by Merck Group (1 mentions)
Dm4000b, by Leica camera (1 mentions)
25 protocols using edu solution
1
Cell Viability and Proliferation Assay
2
EdU Cell Proliferation and Viability Assay
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For EdU cell proliferation assay, cells (approximately 1 × 105 cells/well) were seeded and cultured in 12-well plates. After indicated treatments, cells were incubated with EdU solution (Beyotime Biotechnology, cat. no. C0071S) for 2 h. EdU incorporation was measured according to the manufacturer's instructions. To monitor cell viability, a cell counting kit (CCK-8, Dojido Molecular Technologies, cat. no. DJDB4000X) was used by following the manufacturer's instructions. Briefly, cells (approximately 7.5 × 103/well) were seeded and cultured in 96-well plates. After treatment, cells were washed with PBS twice, and incubated with 100 μL non-phenol red medium containing 10% CCK-8 solution at 37 °C for 1-4 h in each well. The absorbance was measured at 450 nm using an automatic microplate reader (Synergy H1, Bio-tek, Winooski, VT, USA).
3
Cell Viability, Proliferation, and Colony Assays
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Cell counting kit‐8 (CCK‐8) assay was utilized for examining cell viability. Shortly, transfected tumor cells (2000 cells per well) were mixed with CCK‐8 reagent (10 μl, Beyotime, Jiangsu, China) at indicated time points. After being incubated for 3 h, absorbance was detected according to a microplate reader. The experiment was conducted thrice.
For the 5‐ethynyl‐2′‐deoxyuridine (EdU) assay, transfected SCL‐1 and A431 cells in 24‐well plates were reacted with 20 μM of EdU solution (Beyotime) for 2 h, followed by a 4% paraformaldehyde fixture. After being added with Click Additive Solution, cells were subjected to staining nucleic acids using 4′,6‐diamidino‐2‐phenylindole. Under a fluorescence microscope (×100; Leica, Wetzlar, Germany), EdU‐positive cells were analyzed. This assay was carried out at least three times.
In addition, tumor cells in six‐well plates were cultured 14 days later. The generated colonies (cell mass containing > 50 cells) were fixed using methanol (Sangon Biotech, Shanghai, China) and stained with Giemsa (Beyotime), followed by calculating using a microscope. Colony formation assay was conducted at least three times.
For the 5‐ethynyl‐2′‐deoxyuridine (EdU) assay, transfected SCL‐1 and A431 cells in 24‐well plates were reacted with 20 μM of EdU solution (Beyotime) for 2 h, followed by a 4% paraformaldehyde fixture. After being added with Click Additive Solution, cells were subjected to staining nucleic acids using 4′,6‐diamidino‐2‐phenylindole. Under a fluorescence microscope (×100; Leica, Wetzlar, Germany), EdU‐positive cells were analyzed. This assay was carried out at least three times.
In addition, tumor cells in six‐well plates were cultured 14 days later. The generated colonies (cell mass containing > 50 cells) were fixed using methanol (Sangon Biotech, Shanghai, China) and stained with Giemsa (Beyotime), followed by calculating using a microscope. Colony formation assay was conducted at least three times.
4
Proliferation Assay of U-251 MG and U-87 MG Cells
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U-251 MG and U-87 MG cells were tripsinized after a 24 h of transfection and then seeded into 96-well plates at a density of 2 × 104 per well. Twelve hours later, the cells were labeled with 10 μM EdU solution (Beyotime, Shanghai, China) at 37 °C for 3 h. After fixation with 4% methanal in PBS at 28 °C for 20 min and subsequent permeation with 0.5% Triton X-100 (Sangon Biotech) in PBS at 28 °C for 10 min, the cells were incubated with 50 µL of 1 × Click Additive Solution (Beyotime) at 28 °C for 30 min. The nucleus was stained with 5 μg/mL DAPI (Yeasen). Finally, the fluorescent dots were observed and photographed using a fluorescence microscope (MOTIC, Hongkong, China) and then photographed. Image J v1.48u was used to count the number of cells.
5
HUVEC Proliferation Assay with PUMLSC-sEVs
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HUVECs were seeded in six-well plates and cultured for 12 h, added to medium containing PUMSLC-sEVs, cultured for 12 h, treated with EdU solution (Beyotime, China) for 2 h, and incubated in the click reaction solution for 30 min at 37 °C in the dark. Cells were stained according to the kit instructions and then imaged under a fluorescence microscope, and the percentage of EdU-positive cells was calculated.
6
Cell Proliferation Analysis via EdU Assay
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Cells from six‐well plates were digested with trypsin and seeded into 96‐well plates at a density of 4 × 104 cells per well after 48 h of transfection. EdU solution (Beyotime, China) was diluted at a ratio of 1000:1 to prepare EdU culture medium, which was added to each well (100 μL per well). After 2 h of incubation, excess EdU medium was removed, and cells were washed with PBS. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X‐100 (Dow Chemical Company, USA), stained with Hoechst 33342 (Beyotime, China), and observed under a fluorescence microscope after incubation.
7
Measuring MC Proliferation with EdU and PCNA
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To determine the proliferation level of MCs, we performed the 5-ethynyl-2′-deoxyuridine (EdU) labeling assay and observed the expression level of PCNA using immunofluorescence. The incorporation of EdU was observed with a Zeiss Axio Observer Z1 microscope (Carl Zeiss, Thornwood, NY, USA) after the MCs were incubated with EdU solution (Beyotime Biotechnology) according to the manufacturer’s instructions. For the staining of PCNA, the MCs were fixed in 4% paraformaldehyde solution and permeabilized with 0.5% Triton X-100 for 15 min. After blocked by 5% BSA, the cells were incubated with anti-PCNA antibody (#13-3900, 1:100, ThermoFisher Scientific) and corresponding secondary antibody (ThermoFisher Scientific) and 4′,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology). The samples were observed with a Carl Zeiss microscope.
8
Cell Proliferation Assay with EdU
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The cells were seeded on 96-well plates with a density of 1 × 105 cells in each well. EdU solution (Beyotime, China) was diluted with culture medium in a ratio of 1000:1. The amount of 50 μM EdU solution and 100 μM culture medium were applied to each well after transfection and incubated for 2 h. The 4′,6-diamidino-2phenylindole (DAPI, Sigma, USA) was added to well plates to stain the cells. Finally, the results were perceived and photographed under a fluorescence microscope.
9
Chondrocyte Proliferation Assay
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Chondrocytes of each group were seeded in a 24-well plate at a density of about 105 cells/ml; 200 μl of 0.1% EdU solution (Beyotime, China, C0078S) was added in each well. After incubated for 4 hours at 37°C, chondrocytes were fixed with 4% paraformaldehyde for 15 minutes, and then infiltrated by 0.3% Triton X-100 (Biosharp, China, BS084) for 15 minutes. Then cells were incubated with Click solution for 30 minutes at 37°C in the dark. After being washed with PBS solution for 3 times, cells were incubated with 0.1% Hoechst 33342 for 10 minutes. Images were obtained under a fluorescence microscope (Leica DM 4000 B, Germany).
10
Cell Proliferation Measurement with EdU
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On the day before the experiment, PC cells (5 × 105 cells/well) were seeded in 24-well plates and then incubated with 10 μM EdU solution (Beyotime, Cat No. C0071S) for 2 h. Cells were fixed with 4% paraformaldehyde and washed with PBS, then permeabilized with 0.3% Triton X-100 and stained using Hoechst and Apollo reaction mixtures.
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