Beyoclick edu 488 cell proliferation detection kit

After being cultured for 3 d, 1.7 × 10⁴ THP‐1 cells from the TCP, T+US, β‐PVDF, and β+US groups were scraped and seeded in the upper chambers of a 24‐well Transwell system. A total of 2 × 10³ tumor cells were seeded in the lower chambers, and monocultured tumor cells were used as the CTR control group. On day 3, the proliferation of tumor cells was evaluated using the BeyoClick EdU‐488 Cell Proliferation Detection Kit (Beyotime, Jiangsu, China) according to the manufacturer's protocol. The percentage was determined across five randomly selected view‐fields of tested samples.

Firstly, we seeded 143B and HOS cells in 6-well plates with a suitable density. After adherence, OS cells were starved for about 8 h with FBS-free DMEM to synchronise cell cycle of each wells. Then cells were transfected with specific siRNA or plasmid. After that, the medium of each wells were replaced with fresh DMEM containing 10 μM EdU and cultured at 37 °C, with 5% CO₂ for another 2 h. Then 4% paraformaldehyde was used to fix the cells for 15 min, and the fixed cells received 20 min of incubation with 0.3% Triton-X-100. Afterwards, cells were cultured with Click reaction buffer for 25 min away from light. Then Hoechst dye 33342 was employed to stain the nuclei. We calculate the cellular proliferation rate in terms of the instructions of the manufacturers (BeyoClick™EdU-488 Cell Proliferation Detection Kit, Beyotime Biotechnology).

The following cell lines, antibodies, and chemicals were employed in this research: GES-1, AGS, MKN7, SGC7901, NCI-N87 (Cell Bank of Type Culture Collection of the Chinese Academy of Sciences), anti-NPR3 (Abcam, A19038), anti-GAPDH (Proteintech, Cat No: 10494–1-AP), anti-E-cadherin (Proteintech, Cat No: 60335–1-Ig), anti-N-cadherin (Proteintech, Cat No: 22018–1-AP), anti-Vimentin (Proteintech, Cat No: 10366–1-AP), HRP Goat Anti Rabbit IgG (H+L) (Abclonal, AS014), Crystal violet (Aladdin, C110703), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT, Aladdin: M158055) and BeyoClick ™ EdU-488 Cell Proliferation Detection Kit (Beyotime, C0071S).

The 5-ethynyl-2-deoxyuridine (EDU) assay was performed according to the manufacturer specification of BeyoClick™ EDU-488 cell proliferation detection kit (Beyotime Biotechnology, Hangzhou, China). Briefly, glioma cells cultured in a confocal dish, after transfection, and an EDU reagent were added and incubated for 2 h. After washing with phosphate-buffered saline (PBS) twice, the cells were fixed with 4% paraformaldehyde for 30 min. Then, the cells were incubated with Apollo staining reaction liquid for 30 min to detect the positive cells. Following staining with 4,6-diamino-2-phenylindole to identify the nucleus, immunofluorescence was observed through a fluorescence microscope at 488 nm.

Firstly, 143B and HOS cells were seeded in 6-well plates with a suitable density. After adherence for 24 hours, the cells were starved for about 8 hours with FBS-free DMEM to synchronize cell cycle of each wells. Then cells were exposed to 0, 8, 16 or 32 μmol/L ML323 for 48h. After that, the medium of each well were replaced with fresh DMEM containing 10 μM EdU and incubated at 37 °C, with 5% CO₂ for another 2 hours. Then fixed with 4% paraformaldehyde for 15 min and incubated with 0.3% Triton-X-100 for 15 min. Afterwards, the fixed cells were incubated with Click reaction buffer for 30 min away from light. Subsequently, the nuclei were stained with Hoechst dye 33342. Cellular proliferation rate was calculated in terms of the manufacturer's instructions (BeyoClick™EdU-488 Cell Proliferation Detection Kit, Beyotime Biotechnology, Shanghai, China).

The cell proliferation assay of LINC02428 was performed using the Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays. For the CCK-8 assay, cells were planted in 96-well plates at a density of 1000 cells per well. The CCK-8 reagent was purchased from Beyotime (Shanghai, China) and was added for 1, 2, 3, 4, and 5 days. After incubation for 3 h, the absorbance at 450 nm was detected using a microplate reader (BioTek ELx800 Absorbance Microplate Readers). For the EdU assay, BeyoClick™ EDU-488 Cell Proliferation Detection Kit was purchased from Beyotime (Shanghai, China). Cells with a density of ~1 × 10⁴ cells per well were seeded in a 96-well plate. The cultured cells were subjected to EdU labeling, fixation, washing, and nuclear staining as per the manufacturer’s instructions. Finally, the green fluorescence intensity and cell number were observed under a fluorescent microscope.