The stably infected cells of interest were seeded in 96-well plates at 4 × 104 cells per well and cultured to the logarithmic growth phase. The cells were treated with EdU solution, Apollo staining reaction solution, and Hoechst33342 reaction solution (Beyotime Biotechnology, Shanghai, China). Cells were observed under an inverted fluorescence microscope, and images were obtained.
Hoechst 33342 reaction solution
Manufactured by Beyotime
Sourced in China
Hoechst 33342 reaction solution is a fluorescent dye used for staining and visualizing nucleic acids, primarily DNA, in various biological and laboratory applications. It binds to the minor groove of DNA, emitting a blue fluorescence when excited by ultraviolet light. The solution provides a stable and consistent dye concentration for consistent and reliable results.
Automatically generated - may contain errors
5 protocols using hoechst 33342 reaction solution
1
Cell Proliferation Assay with EdU
2
Quantifying Cell Proliferation using EdU Assay
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EdU solution was added to the cell culture plate and incubated for 2 h at room temperature. The cells were then fixed with 40 g/L paraformaldehyde for 30 min, incubated with glycine solution for 8 min and then washed with phosphate-buffered saline (PBS) containing 0.5% Triton X-100. After that, Apollo® staining reaction solution was added and incubated for 30 min at room temperature in the dark. Methanol and PBS were then used to wash the cells 2 times, respectively. Finally, the cells were incubated with Hoechst 33342 reaction solution (C1022, Beyotime Biotechnology Co., Shanghai, China) at room temperature for 20 min in the dark. Under the fluorescence microscope, the red-stained cells (EdU-positive) were the proliferating cells and the blue-stained cells (Hoechst 33342-positive) were the total cells. Three fields of view were randomly selected under 400-fold fields of view. Cell proliferation rate = the number of proliferating cells/the number of total cells ×100%.
3
Hoechst 33,342 Nuclear Staining Protocol
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The cells were treated as described in Section 4.6.1 . Hoechst 33,342 reagent was diluted, and an appropriate volume of 1× Hoechst 33,342 reaction solution (Beyotime Biotechnology, Shanghai, China) was prepared and protected from light. Hoechst 33,342 reaction solution (800 μL, 1×) was added to each well, then culture plates were placed on an orbital shaker and incubated for 30 min at room temperature, protected from light. Hoechst 33,342 was then aspirated, and cells were washed 1–3 times for 10 min each with PBS. Image acquisition and analysis were conducted as described in Section 4.6.1 . The maximum excitation wavelength of Hoechst 33,342 was 350 nm, and the maximum emission wavelength was 461 nm.
4
Cell Viability and Proliferation Assays
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For the CCK-8 assay, 3 × 103 cells were seeded into 100 μl of complete culture media in 96-well plates for various time periods. A CCK-8 kit (Dojindo Laboratories, Japan) was then used to measure cell viability in accordance with the manufacturer’s instructions. The EdU assay was carried out by using a Cell-Light EdU Apollo567 In Vitro Kit (Ribobio, Guangzhou, China) in accordance with the manufacturer’s instructions. In brief, the cells were incubated with 50 μM of EdU solution for 2 h at room temperature. Following fixation with paraformaldehyde and immersion in glycine solution, the cells were permeabilized with 0.5% Triton X-100 in PBS. Next, the cells were stained at room temperature for 30 min in the dark with Apollo staining solution and Hoechst 33342 reaction solution (Beyotime, Shanghai, China). Finally, cells were washed twice with methanol and PBS. Finally, representative images were acquired by a Leica DMI8 microscope.
5
EdU Proliferation Assay Protocol
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The cells were incubated with 50 uM EdU solution (EdU solution and cell medium were mixed at a ratio of 1: 1000) for 2 h at room temperature. After fixation with 40 g/L paraformaldehyde for 30 min, the cells were immersed in glycine solution for 8 min. After that, the cells were permeabilized with 0.5% Triton X-100 in PBS. The cells were then stained with Apollo® staining solution at room temperature for 30 min in the dark, and washed twice with methanol and PBS. Finally, Hoechst 33342 reaction solution (C1022, Beyotime Biotechnology Co., Ltd., Shanghai, China) was used for staining at room temperature for 20 min in the dark [44 (link)]. Three fields of view were randomly selected, and total number of EdU-stained cells, which indicates proliferating cells and Hoechst 33342-stained cells which indicates the total number of cells, were counted. Cell viability was calculated using the following formula: proliferating cells / total cells × 100%. The experiment was repeated three times.
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