Cell light edu cell proliferation detection kit

This assay was analyzed using a Cell-Light™ EdU Cell Proliferation Detection kit (RiboBio, China) according to according to previous methods [50 (link)]. Control or lncRNA-SVUGP2-overexpressing HepG2 and Hep3B stable cell lines were seeded at 1.5 × 10⁵ cells/well in 24-well plates. Then, cells were labeled with 5-ethynyl-2′-deoxyuridine (EdU), which incorporates into actively proliferating cells, and fixed with paraformaldehyde. After DNA staining, cellular immunostaining was observed using an epifluorescence microscope (Leica, Germany). Digital images were acquired and analyzed with ImageJ software.

Each group of isolated tumour cells was seeded onto 96-well plates in triplicate at a density of 10³/well and incubated for 48 h. Subsequently, the cells were incubated for an additional 2 h in the respective media containing 50 μM EdU (RiboBio, Guangzhou, China). Cell proliferation was detected using a Cell-Light™ EdU Cell Proliferation Detection Kit (RiboBio, China) following the manufacturer’s instructions. DNA was incubated with Hoechst 33342 stain (100 μl/well) for 30 min and visualised using an inverted fluorescence microscope (Leica DM5500, Germany). For each EdU experiment, five random fields were imaged at 100 × magnification. Captured images were processed and analysed using ImageJ software. The number of EdU-positive cells was determined by Hoechst nuclear staining and expressed as a percentage of the total number of cells in each field.

The effects of miR-22 on the viability of NSCLC cells were measured using cell counting kit-8 assays (CCK-8; Dojindo, Japan). Cells were planted in 96-well plates and were allowed to adhere overnight. After transfection of miR-22 mimics or inhibitors, CCK-8 reagent was added and incubated for 1 h at 37°C, and the absorbance was performed at 450 nm using a spectrophotometer (Bio-Rad, USA).
Cells were seeded in 24-well plates and were allowed to adhere overnight in complete medium. After 48 h of transfection with the miR-22 mimics and their negative control, cells were incubated with EdU (5-ethynyl-2-deoxyuridine) for 8 hours before staining. The actively proliferating cells were then detected using a CellLight™ EdU Cell Proliferation Detection Kit (RiboBio, China) following the manufacturer's instructions. Percentage of proliferative cells was observed with an epifluorescence microscope (Leica, Germany).

Cell proliferation was determined by CCK-8 assay and EdU (5-ethynyl-20-deoxyuridine) staining. For the CCK-8 assay, 1000 cells of different groups/well were seeded into 96-well plates. At each time point (24, 48, 72, and 96 h after seeding), 10 μL of CCK-8 reagent (Beyotime Biotech, Shanghai, China), and 100 μL of medium were added to each well followed by incubation at 37°C for 2 h. The optical density of the wells was measured at 450 nm using a microplate reader (SpectraMax i3, Molecular Devices, San Jose, CA, USA). Data were from three separate experiments, each with three replications. For the EdU staining assay, the Cell-Light™ EdU Cell Proliferation Detection Kit (RiboBio) was used. Cells from different groups were seeded into 96-well plates at the appropriate density. After incubation at 37°C and 5% CO₂ for 48 h, 50 mM EdU reagent was added to the cells, followed by incubation for another 2 h. The cells were then fixed in 4% paraformaldehyde and stained with Apollo Dye solution to label proliferating cells. Nuclei were counterstained with DAPI. The cell proliferation rate was calculated according to the manufacturer’s instructions. Images were obtained using a fluorescence microscope (Zeiss, Jena, Germany).

IPEC-J2 cells were seeded in 96-well plates (Corning Incorporated, Corning, NY, USA) with a density of 1.5 × 10⁵ cells/mL (100 μL per well). After 24 h of incubation, cells were treated with 50 μM CDCA for another 24 h. Cells were incubated with 5-ethynyl-2′-deoxyuridine (EdU) for 2 h before staining. Cell proliferation was detected using Cell-Light EdU Cell Proliferation Detection Kit (Ribobio, Guangzhou, China) according to the manufacturer’s protocol. The percentage of proliferative cells was determined by quantitation of EdU-positive cells using an Olympus fluorescent microscope (Olympus, Tokyo, Japan).