According to the manufacturers’ instruction, the 2 × 104 cells TE-1 and KYSE150 cells were seeded in 24-well plates to determine the number of proliferation cells with an Edu kit (BeyoClick™ EdU-488, Beyotime).
Beyoclick edu 488
Manufactured by Beyotime
Sourced in China
BeyoClick™ EdU-488 is a fluorescent nucleoside analog that can be incorporated into newly synthesized DNA. It is used for the detection and visualization of DNA replication in proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Cell counting kit 8 (cck8), by Beyotime (2 mentions)
Glomax multi detection system, by Promega (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Bx63f microscope, by Olympus (1 mentions)
U rfl t, by Olympus (1 mentions)
48 well plate, by Corning (1 mentions)
Cell counting kit 8 (cck8), by Sangon (1 mentions)
Ab32084, by Abcam (1 mentions)
Ab179467, by Abcam (1 mentions)
Aggrecan, by Proteintech (1 mentions)
Anti rabbit secondary antibody, by Abcam (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Ab150077, by Abcam (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
12 protocols using beyoclick edu 488
1
Cell Proliferation Assay with EdU-488 Kit
2
HT22 Cell Proliferation Assay
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The HT22 cells were seeded in 96-well or 24-well plates and incubated with the extract of NT-4 silkworm cocoon or WT silkworm cocoon at a dosage of 100 ng/ml for 24 h. Cell proliferation of HT-22 was measured using the Cell Counting Kit-8 (Beyotime, Shanghai, China) and the BeyoClick™ EdU-488 (Beyotime, Shanghai, China) according to the manufacturers’ protocol. For the CCK-8 assays, HT22 cells treated by different extracts were incubated with CCK-8 solution (10 μL per well of a 96-well plate) at 37°C for 1 h, and the absorbance was measured at 450 nm in a Glomax Multi Detection System (Promega, Madison, WI, United States). For the EdU staining, HT22 cells after treatment were labeled by EdU at a concentration of 2 μM at 37°C for 2 h. The labeled cells were immediately fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) in PBS and permeabilized by 0.5% Triton® X-100 (Beyotime, Shanghai, China), followed by EdU detection using Alexa Fluor 488 and the proliferated cells were photographed under a fluorescence microscope (Olympus, Tokyo, Japan).
3
Quantifying Cell Proliferation with EdU Assay
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The EdU cell proliferation assay was performed using BeyoClick™ EdU-488 (Beyotime, C0071S) to assess cell proliferation. EdU solution was added to the cell culture medium to a final concentration of 10 μM 2 hours prior to harvesting. Cells were fixed with 4% paraformaldehyde for 15 minutes and washed again with PBS. After permeabilization with 0.3% Triton X-100 and further washing with PBS, Click Additive Solution was added and incubated for 30 minutes at room temperature. Cells were washed with PBS, and Hoechst was added for incubation at room temperature for 10 minutes. Finally, cells were washed three times with PBS and imaged using Olympus BX63F microscope with a CCD camera.
4
EdU Proliferation Assay for DNA Synthesis
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The EdU proliferation assay was conducted to detect DNA synthesis. Briefly, cells were seeded in 24-well plates at the density of 1 × 104 cells per well. After 24 h, the cells were incubated with 5-ethynyl-2'-deoxyuridine (EdU) (BeyoClickEdU 488, Beyotime, C0071l) for 2 h. After fixed, the labeled cells were incubated in the click addictive solution in a dark humidifier for 30 min. The labeled cells glow green and the nuclei stained by Hoechst 33342 glow blue. Images were taken by fluorescence microscope (Olympus U-RFL-T, Tokyo, Japan).
5
Cell Proliferation Assays on MHCC-97H Cells
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For the CCK-8 assay, MHCC-97H cells were plated at 2 × 103 cells per well in 96-well plates and incubated overnight in DMEM supplemented with 10% FBS. The cell proliferation index was measured using a Cell Counting Kit-8 (Beyotime, Shanghai, China) at 0, 24, 48, and 72 h post-transfection according to the manufacturer’s instructions. The absorbance was measured at a wavelength of 450 nm.
For the EdU assay, MHCC-97H cells were seeded into 48-well plates (Corning). When the confluence of MHCC-97H cells reached 80%, BeyoClick™ EdU-488 (Beyotime, Shanghai, China) was used to determine the proliferation rate of the cells. After staining, the cells were imaged using a confocal laser-scanning microscope (ZEISS, Germany).
For the EdU assay, MHCC-97H cells were seeded into 48-well plates (Corning). When the confluence of MHCC-97H cells reached 80%, BeyoClick™ EdU-488 (Beyotime, Shanghai, China) was used to determine the proliferation rate of the cells. After staining, the cells were imaged using a confocal laser-scanning microscope (ZEISS, Germany).
6
Cell Viability and Proliferation Assays
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Cell viability and proliferation were determined with a Cell Counting Kit-8 (E606335, Sangon Biotech) and a BeyoClick EdU-488 (C0071S, Beyotime). For the CCK-8 assay, SW780 or MGHU3 cells were transfected with the indicated siRNAs, then seeded into 96-well plates at a density of 1,000 cells/well. According to the manufacturer's recommendations, 10 μL of CCK-8 solution was added into the wells and the mixture was cultured for 3 hours. The absorbance was recorded at 450 nm.
For the EdU assay, cells were cultured in 24-well plates and transfected with the indicated siRNAs. At 24 hours post-transfection, cells were incubated with 50 μmol/L EdU for 2 hours at 37°C and then fixed in 4% formaldehyde for 30 minutes. Glycine was added to neutralize the formaldehyde. After permeabilizing with 0.3% TritonX-100 for 10 minutes at room temperature, 1 × Click reaction cocktail (100 μL) was used to react with the EdU for 30 minutes. Finally, 1 × Hoechst (100 μL) was used to stain the nuclei. Cells were visualized using a Leica DM750 microscope.
For the EdU assay, cells were cultured in 24-well plates and transfected with the indicated siRNAs. At 24 hours post-transfection, cells were incubated with 50 μmol/L EdU for 2 hours at 37°C and then fixed in 4% formaldehyde for 30 minutes. Glycine was added to neutralize the formaldehyde. After permeabilizing with 0.3% TritonX-100 for 10 minutes at room temperature, 1 × Click reaction cocktail (100 μL) was used to react with the EdU for 30 minutes. Finally, 1 × Hoechst (100 μL) was used to stain the nuclei. Cells were visualized using a Leica DM750 microscope.
7
Mesenchymal Stem Cell Chondrogenic Differentiation
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ASA VI was purchased from Chengdu Must Bio-Technology Co. Ltd. (purity> 99%, China). Mesenchymal stem cell medium (MSCm) (7501), fetal bovine serum (FBS, 7552) and Dulbecco’s phosphate-buffered saline (DPBS, 0303) were purchased from ScienCell (USA). DMEM/F12 was purchased from Gibco (21,041,025, USA). BeyoClick EdU-488 was purchased from Beyotime Institute of Bio-Technology Co. Ltd. (Beyotime, C0071S, China). ProtoScript II cDNA was purchased from NEB (m3003L, USA). DAPI (D9542), dimethylmethylene blue (DMMB, 341088), glycine (410225), glacial acetic acid (S7653) and bovine chondroitin 4-sulfate as standard (C9819) were purchased from Sigma-Aldrich (USA). Primary antibodies for β-catenin (ab179467) and paxillin 1 (PAX1, ab32084) were purchased from Abcam (USA). Aggrecan was purchased from Proteintech (13880–1-AP, USA), and smad2/3 (8685 T), p-smad2/3 (8828S), ERK1/2 (4695 T), and p-ERK1/2 (4370 T) were purchased from Cell Signaling (USA). Anti-rabbit secondary antibodies were purchased from Abcam (ab150077, USA).
8
Cell Proliferation Assay via EdU Labeling
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Twenty-two hours after performing cell treatment experiments, cells were labeled with EdU for 4 h and then fixed to stain the cells using the EdU staining kit BeyoClick™ EdU-488 (Cat: C0071S, Beyotime, Shanghai, China). The EdU-positive cells were then observed and photographed under a fluorescence microscope (MOTIC, Hongkong, China). The number of cells were counted using Image J 1.52v (NIH, Bethesda, MD, USA).
9
Senescence Assay with Exendin-4 Pretreatment
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MSCs and Min6 were cultured on 12-well plates with a total number of 1 × 104 and 1 × 105 cells per well respectively. Cells were pretreated with Exendin-4 peptides or CM as same as the senescence assay. Cells were stained with BeyoClick EdU-488 (C0071S, Beyotime) for 2 hours according to the manufacturer’s instruction, and fluorescence was measured at 488 and 346 nm using fluorescent microscopy.
10
Cell Proliferation Assays for Cal27 Cells
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Cell counting kit-8 (CCK-8; GLPBIO, Montclair, USA) was used to detect cell proliferation. After 18–24 h of transfection, Cal27 cells were seeded onto 96-well plates (4 × 103 cells/well). After 24, 48, and 72 h of culture, 100 µL of fresh medium containing 10% CCK-8 reagent was added to each well, and then, cells were cultured at 37 °C in 5% CO2 for 2 h. The optical density at 450 nm was detected with a microplate reader (BioTek Instruments, Winooski, USA).
Moreover, cell proliferation was detected through the 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay. After 18–24 h of transfection, EdU was added to the 6-well plates, and the cells were cultured for an extra 2 h. Next, the proliferation rate of the cells was assessed with BeyoClick™ EDU-488 (Beyotime, Shanghai, China) following the manufacturer’s instructions. Five randomly selected regions were examined with a fluorescence microscope (Olympus, Tokyo, Japan) to analyze the proliferation rate.
Moreover, cell proliferation was detected through the 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay. After 18–24 h of transfection, EdU was added to the 6-well plates, and the cells were cultured for an extra 2 h. Next, the proliferation rate of the cells was assessed with BeyoClick™ EDU-488 (Beyotime, Shanghai, China) following the manufacturer’s instructions. Five randomly selected regions were examined with a fluorescence microscope (Olympus, Tokyo, Japan) to analyze the proliferation rate.
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