Beyoclick edu 594 cell proliferation detection kit

Cell proliferation was evaluated using 5-ethynyl-2-deoxyuridine (EdU) labeling and cell counting assays. The BeyoClick™ EdU-594 Cell Proliferation Detection Kit (Cat#C0078S, Beyotime, China) was used. Transfected hPASMCs were plated in six-well plates for 36 h, and the preheated EdU working solution was added to each well. After incubation for 2 h, EdU-labeled cells were fixed with 4% paraformaldehyde for 20 min and subsequently incubated with 0.3% TritonX-100 for 20 min and click reaction solution for 30 min. Hoechst 33,342 solution was then added to label the nuclei of living cells. Images were acquired using fluorescence microscopy.
For the cell count assay, transduced hPASMCs were plated at a density of 2.5 × 10⁵ cells per well in a six-well plate. After incubation for 36 h, cells were harvested with trypsin–EDTA, and counted using 0.4% trypan blue with a hemocytometer.

Chondrocytes were divided into four groups including (1) control, (2) IL-1β + PBS, (3) IL-1β + EVs, and (4) IL-1β + LPS-pre EVs groups. Chondrocytes were seeded into 24-well plates and after chondrocytes of each group reached 70–80% confluence, IL-1β + PBS, IL-1β + EVs, and IL-1β + LPS-pre EVs groups were cultured with IL-1β (10 ng/ml) (Proteintech)-containing medium for 12 h. The medium for the control group was replaced with complete culture medium containing EdU (10 μM). The medium of the IL-1β + PBS group was replaced with complete culture medium containing IL-1β (10 ng/ml), PBS (100 μl), and EdU (10 μM). The medium for third group was replaced with complete culture medium containing IL-1β (10 ng/ml), EVs (10¹⁰ particles/ml), and EdU (10 μM), and the medium of the IL-1β + LPS-pre EVs group was replaced with complete culture medium containing IL-1β (10 ng/ml), LPS-pre EVs (10¹⁰ particles/ml), and EdU (10 μM). Extracellular vesicles-free serum was used in all the above complete media. Cells were incubated for 10 h. After incubation, analysis was conducted using BeyoClick™EdU-594 Cell Proliferation Detection Kit (Beyotime) following the manufacturer’s instructions. The nuclei of the proliferating cells emitted red fluorescence under excitation light of the fluorescence microscope. Cell proliferation of each group was observed and recorded using fluorescence microscope.

EdU kit was used to test chondrocyte proliferative capability. A cell co-culture system was created by employing a 24-well transwell chamber (pore size: 0.4 μm, Corning) to evaluate chondrocyte proliferative potential. First, the chondrocytes were seeded into 24-well plates and cultured to around 90% cell density for future use. THP-1 cells were seeded in the transwell upper chamber followed by using lipopolysaccharide (LPS) and Nigericin (Nig) treatment after cell attachment with phorbol myristate acetate (PMA, P1585, Sigma-Aldrich) to stimulate THP-1 cells to differentiate into macrophages. The co-culture system was then established by inserting transwell chambers into chondrocyte-adherent 24-well plates and incubating for 24 h in a medium containing EdU (10 μM). The proliferation rate was evaluated using BeyoClick™EdU-594 Cell Proliferation Detection Kit (Beyotime) according to manufacturer's instructions. Fluorescence microscopy and flow cytometry were used to examine cell proliferation in each group.

The proliferation of ovarian cells HEY was investigated with BeyoClick™ EdU-594 Cell Proliferation Detection Kit (Beyotime, C0078S, Shanghai, China) according to the manufacturer’s protocol. Cells were seeded into 6-well plates overnight at 37 °C. After treatment with or without 20 uM ML355 for 8 h, HEY cells were incubated with EdU working solution (10 uM) for 2 h. Then, cells were washed with PBS twice and fixed with 4% paraformaldehyde for 15 min at room temperature. Next, the cells were washed with PBS (Servicebio, G0002, Wuhan, China) containing 3% BSA (Aladdin, 9048-46-8, Shanghai, China). The cells were incubated with PBS containing 0.3% Triton X-100 (Sigma Aldrich, 9036-19-5, Shanghai, China) for 12 min at room temperature, and were then washed twice with PBS containing 3% BSA. Finally, cells were incubated with Click Additive Solution for 30 min in the dark at room temperature, and subsequently stained with 1× Hoechst for nucleus staining. Images were captured with a fluorescence microscope (Leica, Weztlar, Germany). Cells at the DNA replication phase emitted red fluorescence, while the cell nuclei emitted blue fluorescence.

Cellular proliferation was determined utilizing BeyoClick™ EdU-594 cell proliferation detection kit (C0078S; Beyotime, Shanghai, China). The transfected cells were inoculated onto 24-well plates (8 × 10⁵ cells/well). All operations were carried out following the instructions. Under a fluorescence microscopy, the images were acquired and analyzed.