To detect effects of compounds on cell proliferation is one of basic method to evaluate the antitumor activity of drug effect. It is widely accepted that the most accurate way is to directly detect the synthesis of DNA in cells. EdU (5-ethynyl-2′-deoxyuridine) is a thymidine analog which can be substituted for thymidine in DNA synthesis. We used the EdU-594 cell proliferation detection kit (C0078L, Beyotime, China) to examine the synthesis of DNA according to manufacturer’s instruction. The results of the EdU staining were photographed under a fluorescence microscope.
Edu 594 cell proliferation detection kit
Manufactured by Beyotime
Sourced in China
The EdU-594 cell proliferation detection kit is a laboratory tool used to measure and analyze cell proliferation. It functions by detecting the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA, providing a quantitative assessment of cell division activity.
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Lab products found in correlation
Fluorescence microscope, by Olympus (2 mentions)
Puerarin, by Maclin Biochemical Technology (1 mentions)
Tunel staining kit, by Wuhan Servicebio Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Affinity Biosciences (1 mentions)
Igg h l alexa fluor 488, by Abcam (1 mentions)
Bcl2 associated x (bax), by Affinity Biosciences (1 mentions)
Cck 8 kit, by MedChemExpress (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Cellinsight cx7 high content screening platform, by Thermo Fisher Scientific (1 mentions)
6 protocols using edu 594 cell proliferation detection kit
1
EdU Proliferation Assay Protocol
2
EdU Proliferation Staining Assay
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EdU staining assay was performed by EdU-594 Cell Proliferation Detection Kit (Beyotime). In brief, cells were pre-cultured in 6-well plates and incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) for 2 h. The cells were stained with Azide 594. The nuclei were then stained with 4'-6-diamidino-2-phenylindole (DAPI) for 10 min.
Finally, EdU-positive cells were observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan).
Finally, EdU-positive cells were observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan).
3
Puerarin Modulates P53 and Apoptosis Pathways
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Puerarin was purchased from Macklin (Art. No. P816259, China) with purity ≥ 98% (HPLC). Antibodies against P53, P21, PTEN, BRCA1, BIRC5, CTGF, Bax, Bcl-2, caspase 3, cleaved caspase 3, GAPDH, Ki67, and PCNA were purchased from Affinity Biosciences (USA); IgG H&L (Alexa Fluor® 488) was from Abcam (UK); CD3, CD4, and CD8 were from BD Biosciences Pharmingen (USA). The CCK-8 kit was obtained from MedChemExpress (USA); the cell apoptosis kit was obtained from BD Biosciences Pharmingen (USA); the EdU-594 cell proliferation detection kit was obtained from Beyotime Biotechnology (China); the TUNEL staining kit was obtained from Servicebio (China); ELISA kits for IL-6 and TNF-α were purchased from MEIMIAN (China); the ELISA kit for CA125 was purchased from ZSGB-BIO (China).
4
Measuring HepG2 Cell Proliferation
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HepG2 cells (5×104 cells/well) were seeded in 24-well plates and treated with or without 100 ng/ml EGF for 24 h at 37°C or 100 ng/ml EGF + 3 µM MK-2206 for 24 h at 37°C before being replaced with medium containing EdU (10 µM; EdU-594 cell proliferation detection kit; Beyotime Institute of Biotechnology). The medium was removed after continued incubation for 2 h, and the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed three times with PBS. Triton X-100 was used for permeabilization for 15 min at room temperature, and the slides were washed three times with PBS. Click reaction solution was then added and incubated for 30 min at room temperature in the dark. Hoechst-33342 staining was performed for 10 min at room temperature. The cells were then observed under an inverted fluorescence microscope, images were captured and the cell proliferation rate was calculated according to the red fluorescence ratio.
5
Evaluating ShtIX Effects on NSCLC Proliferation
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To detect the effects of ShtIX on NSCLC cell proliferation, we first used cell clone formation experiments to observe the single cells to form a colony. Briefly, 5×102 cells were seeded in a 6-well cell culture plate. After being inoculated with the corresponding dose of ShtIX, the cells were grown for 12 days (changing the culture medium containing ShtIX every 3 days). Then, the cells were fixed, stained, counted, and photographed. After that, we used the EdU-594 cell proliferation detection kit (Beyotime, Shanghai, China) to assess the DNA synthesis following the manufacturer’s instructions. Cell nuclei were counterstained with DAPI (Sigma-Aldrich, St Louis, United States). The cells incorporating EdU were monitored under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).
6
Evaluating Antitumor Activity via EdU-Based Cell Proliferation
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To detect effects of compounds on cell, proliferation examination is one of basic method to evaluate the antitumor activity. The rate of DNA synthesis can re ect the rate of cell proliferation. EdU (5-ethynyl-2′deoxyuridine) is a thymidine analog which can be substituted for thymidine in DNA synthesis. We used the EdU-594 cell proliferation detection kit (C0078L, Beyotime, China) to examine the synthesis of DNA according to manufacturer's instruction. The results of the EdU staining were photographed by CellInsight CX7 High-Content Screening (HCS) platform (Thermo Scienti c, US).
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