Nf κb activation kit

Briefly, 1.0×10⁴ cells were seeded in a 96-well plate and incubated overnight at 37ºC with 5% CO₂. The cells were pretreated with different concentrations of the compound for 3 hours and then stimulated with 1 ng/mL of tumor necrosis factor (TNF)-α for 30 minutes. The medium was removed and the cells were fixed and stained with a nuclear factor kappa B (NF-κB) activation kit (Thermo Scientific) according to the manufacturer’s instructions. The plate was examined on an ArrayScan high-content screening reader. Calculation of the cytoplasmic and nuclear NF-κB intensity ratio was carried out using Cytoplasm to Nucleus Translocation BioApplication software. The average intensity of 200 cells per well was quantified, and the ratios were then compared with TNF-α-stimulated, treated, and untreated cells.47

Briefly, 1×10⁴ PC3 cells were seeded in a 96-well plate and incubated overnight at 37°C with 5% CO₂. The cells were treated with different concentrations of the compound biseugenol B for 3 hours and then stimulated with 10 ng/mL of tumor necrosis factor-alpha (TNF-α) for 30 minutes. Briefly, a number of PC cells (1×10⁴) were seeded and incubated in a 96-well plate with 5% CO₂ at 37°C. The treatment of the cells with various concentrations of biseugenol B compound was carried out for 180 minutes, followed by stimulation with 10 ng/mL of TNF-α for half an hour. Later, eliminating the medium and fixing the cells, which was followed by staining them with NF-κB activation kit (Thermo Fisher Scientific, Waltham, MA, USA), were carried out based on the manufacturer’s protocol. On an ArrayScan High-Content Screening Reader, the plate was analyzed. Measuring the intensity ratio of nuclear NF-κB as well as the cytoplasm NF-κB was performed by Cytoplasm to Nucleus Translocation BioApplication software (Thermo Fisher Scientific). For 200 cells/well, the quantification of the average intensity was done by comparing different ratios of TNF-α stimulated in untreated and treated cells.27 ,37 (link)