DES was purchased from Santa Cruz Biotechnology, Inc. (CA, USA), diluted in DMSO(dimethylsulfoxide) to 500 M and stored at -20°C. The final concentrations used here were 2×10−7, 2×10−6, and 2×10−5 M, and they were freshly diluted with DMEM to their final concentrations. Controls were treated with the same amount of DMSO (0.04%) used in the corresponding experiment. A TRIzol Reagent Kit was obtained from Invitrogen (Carlsbad, CA, USA), and a PrimeScript RT Reagent Kit was purchased from Takara (Otsu, Japan). GoTaq Hot Start Green Master Mix was obtained from Promega (Wisconsin, USA). Dnmt1, Dnmt3a, and Dnmt3b antibodies were obtained from Santa Cruz Biotechnology, Inc. An HRP-conjugated(horseradish peroxidase-conjugated) secondary antibody, an enhanced chemiluminescence kit and an Annexin V–FITC(fluorescein isothiocyanate) Apoptosis Detection Kit were purchased from Beyotime (Shanghai, China). An EDU(5-Ethynyl -2’- deoxyuridine) Cell Proliferation Kit was obtained from Ribo (Guangzhou, China), and an EZ DNA Methylation-Gold Kit was purchased from Zymo Research (Orange, CA, USA).
5 ethynyl 2 deoxyuridine edu cell proliferation kit
Manufactured by RiboBio
Sourced in China
5-ethynyl-2′-deoxyuridine (EdU) cell proliferation kit is a laboratory tool used to detect and quantify cell proliferation. It incorporates an alkyne-modified nucleoside, EdU, into the DNA of actively dividing cells. The incorporated EdU can then be detected using a fluorescent azide-based detection method.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (1 mentions)
Trizol reagent kit, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Gotaq hot start green master mix, by Promega (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by Beyotime (1 mentions)
Dnmt3a, by Santa Cruz Biotechnology (1 mentions)
Dnmt3b, by Santa Cruz Biotechnology (1 mentions)
Dnmt1, by Santa Cruz Biotechnology (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
Cell culture plate, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tgf β2, by Proteintech (1 mentions)
Alexa fluor 555, by Abcam (1 mentions)
α sma, by Proteintech (1 mentions)
Zoledronate, by Novartis (1 mentions)
6 protocols using 5 ethynyl 2 deoxyuridine edu cell proliferation kit
1
Epigenetic Profiling of DES Treatment
2
CD4+ T Cell Proliferation Assay with MSCs
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Mononuclear cells were obtained from spleens using grinding and Ficoll Lymphocyte Separation Medium (Hao Ocean Creatures, China) to isolate lymphocytes according to the manufacturer’s instructions. Then, anti-rat CD4-FITC and separation buffer (Miltenyi Biotec, Germany) were used for the isolation of CD4+ T lymphocytes according to the manufacturer’s instructions. One milliliter of T lymphocytes was seeded in the lower layer of a transwell (concentration 2.5 × 104/ml) with RPMI 1640, 10% FBS, 2.5 μg/ml phytohemagglutinin, and 10 ng/ml IL-2. The differently handled MSCs (MSC, Vector-MSCs, and IL33-MSCs) were seeded in the upper layer of a transwell (3-μm pore size; Costar, USA) and co-cultured for 72 h, and Edu (5-ethynyl-2′-deoxyuridine) cell proliferation kit (RiboBio, China) for proliferation assay was added for another 2 h before T cell proliferation was analyzed using flow cytometry.
3
Zoledronate Inhibits Fibrosis in TGF-β1-Induced Cells
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Zoledronate was kindly provided by Novartis Pharma Stein AG (Stein, Switzerland). Antibodies to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TGF-β1, TGF-β2 and α-SMA were purchased from Proteintech Group (Wuhan, China). Antibodies to Smad2, Smad3, p-Smad2, p-Smad3 and α-SMA (Alexa Fluor® 555) were obtained from Abcam (Shanghai, China). Recombinant human TGF-β1 was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). FBS, DMEM, DAPI, Super Signal West Pico Chemiluminescent Substrate and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The 5-ethynyl-2-deoxyuridine (EdU) cell proliferation kit was purchased from Guangzhou RiboBio (Guangzhou, China). Propidium iodide reagent was obtained from Tianjin Sungene Biotech (Tianjin, China). Cell culture plates and transwell plates with 8-µm pore polycarbonate membranes were purchased from Corning Incorporated (Corning, NY, USA). Rat tail tendon collagen type I (5 mg/mL) in 0.006 N acetic acid was purchased from Shengyou Biotechnology Co., Ltd. (Hangzhou, China).
4
Cell Proliferation and Viability Assay
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The 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation kit (RiboBio, Guangzhou, China. Catalog No. C10310) was used to determine cell proliferation. According to the manufacturer's instructions, EdU was infiltrated into the proliferating cells, and the ratio of proliferating cells was calculated by fluorescence microscopy.
Cell proliferation was also determined by Western blot analysis of proliferating cell nuclear antigen (PCNA). PCNA antibody (Cell Signaling Technology, MA, USA. Catalog No. 13110) and GAPDH antibody (Cell Signaling Technology. Catalog No. 2118) were used in the experiment. GAPDH was used as an internal control.
Cell viability was determined using the CCK8 detection kit (Beyotime, Wuhan, China. Catalog No. C0037) according to the reagent manufacturer's instructions. A microplate reader (iMark™ Microplate Absorbance Reader, Bio-Rad, CA, USA) was used to detect the absorbance of the sample at 450 nm (optical density at 450, OD 450). Cell viability was expressed as a percentage.
Cell proliferation was also determined by Western blot analysis of proliferating cell nuclear antigen (PCNA). PCNA antibody (Cell Signaling Technology, MA, USA. Catalog No. 13110) and GAPDH antibody (Cell Signaling Technology. Catalog No. 2118) were used in the experiment. GAPDH was used as an internal control.
Cell viability was determined using the CCK8 detection kit (Beyotime, Wuhan, China. Catalog No. C0037) according to the reagent manufacturer's instructions. A microplate reader (iMark™ Microplate Absorbance Reader, Bio-Rad, CA, USA) was used to detect the absorbance of the sample at 450 nm (optical density at 450, OD 450). Cell viability was expressed as a percentage.
5
Cell Viability and Proliferation Assay
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A CCK-8 kit (Dojindo Molecular Technologies, Inc.) was used to assess the cell viability at different time points. A 5-Ethynyl-2′-deoxyuridine (EdU) cell proliferation kit (Guangzhou RiboBio Co., Ltd.) was used to identify the EdU positive rate of CRC cells.
6
SC Proliferation Assay using EdU
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SC proliferation was analyzed using a 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation kit (Ribobio). The SCs transfected with the indicated siRNA were seeded into a 96-well culture plate at a density of 1 × 105 cells/well and incubated with 50 µM EdU for 4 hours. Next, the treated SCs were fixed in 4% paraformaldehyde for 30 minutes and permeabilized with Triton X-100 for 15 minutes. Then, the cells were stained with Apollo fluorescent dye, and the cell nuclei were stained with 5 µg/mL Hoechst 33342, for 20 minutes. The ratio of EdU-positive to total cells was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA) from four randomly selected images photographed under a Olympus fluorescence microscope (Shinjuku, Tokyo, Japan). The EdU assay was carried out in triplicate, with three identical wells included for each sample.
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