Cell light edu dna cell proliferation kit

A Cell-Light EdU DNA Cell Proliferation Kit (Beyotime, Shanghai, China) was used to test cell proliferation according to the manufacturer’s protocol. Briefly, cells in the logarithmic growth phase were seeded in 96-well plates, followed by cell fixation, EdU labeling, Apollo staining, and DNA staining. Finally, an Olympus microscope (Olympus, Tokyo, Japan) was used to acquire images.

Cell Counting Kit-8 (CCK-8) assay: transfectants were seeded in 96-well plates at 2,000 cells/well in 100 µl medium. Subsequently, the cells were treated with 10 μl CCK-8 reagent (M4839, ABMOLE, USA) at different time points. Optical density (ODs) was measured at 450 nm wavelength using a microplate reader (51119770DP, ThermoFisher). EdU experiments were performed using a Cell-Light EdU DNA Cell Proliferation Kit (C0075, Beyotime). Following the manufacturer’s protocol, PANC-1/BxPC-3 cells were incubated with 50 mM EdU solution for 2 h and immobilized in paraformaldehyde (4%). After that, the cells were permeabilized with 0.3% Triton (9036–19-5, Sigma-Aldrich) for 10 min and then sequentially stained with Alexa Fluor 555 azide. Subsequently, three non-overlapped visions with evenly distributed cells were selected randomly for capturing at 200 × magnification by a fluorescence microscope (DMi8, Leica, Germany) at same exposure time. The cell counting was performed by ImageJ 1.52, the threshold was set as 10 in dark background model, and analyze particles were set as 30-infinity. Proliferation assays were independently repeated three times.

Cell proliferation was also analyzed with a Cell-Light EdU DNA Cell Proliferation kit (Beyotime Institute of Biotechnology, China). Naive and endocrine-resistant BrCa cells were seeded in 96-well plates (1 × 10⁴ cells/well) and treated with tamoxifen. After incubation for 48 h, 10 mM EdU was added and incubated at 37°C for 2 h. Cells were fixed with 4% paraformaldehyde and stained with azide 594 (30 min, for proliferating cells) and Hoechst 33,342 (10 min, for nuclei) at room temperature. Images were captured by a fluorescence microscope (Nikon, Japan). The percentage of proliferating cells was calculated using ImageJ software (National Institutes of Health, United States).

The Cell-Light EdU DNA Cell Proliferation Kit (Beyotime, China) was used for the EdU assay according to the manufacturer's guidelines. Cells were inoculated into six-well plates. Edu working solution was added to the plates and incubated for 2 h, after which the cells were fixed with 4% paraformaldehyde for 15 min. The Click working solution was added for light-protected incubation, with the further addition of Hoechst 33342 after 30 min. The cells were incubated for 10 min at room temperature with protection from light, and were then evaluated and imaged using a fluorescent microscope (Olympus, Japan).

Cell proliferation was assessed using a CCK-8 Cell Counting kit (Beyotime, Shanghai, China) in a 96-well plate (2 × 10³ cells per well). A total of 10 µL of CCK-8 solution was added to 100 µL cell suspension and incubated for 2 h, followed by absorbance assessment at 450 nm (n = 5). All experiments were performed in triplicates for each transfection.
The Cell-Light EdU DNA cell proliferation kit (Beyotime, Beijing, China) was used to determine the proliferation rate of the cells. Briefly, the cells were incubated with 50 μM EdU for 2 h before fixation, permeabilization, and EdU staining. The cell nuclei were stained with DAPI at a concentration of 5 μg/mL for 30 min, and the cells were examined using a florescence microscope (Olympus, Tokyo, Japan).
Cell cycle was determined by flow cytometry, as described previously [42 (link)]. Treated cells were harvested and washed two times with cold PBS and then fixed with 1 mL of 70% ethanol overnight at 4 °C. Fixed cells were centrifuged for 3 min at 1200 g and then washed with cold PBS and resuspended in PBS with 50 mg/mL prepodium iodide (PI) and 1 mg/mL RNase. The stained cells were analyzed for DNA content by fluorescence-activated cell sorting (FACs) in a FACs Calibur (Becton Dickinson Instrument, San Jose, CA, USA). Cell cycle fractions were quantified using the CytExpert 2.0 software (Becton Dickinson).