Briefly, 1.0×104 MDA-MB-231 cells were seeded in a 96-well plate and incubated overnight at 37°C in 5% CO2. The cells were pretreated with different concentrations of boldine for 3 hours and then stimulated with 1 ng/mL tumor necrosis factor alpha (TNF-α) for 30 minutes. The medium was removed and the cells were fixed and stained with nuclear factor kappa B (NF-κB) activation kit from Thermo Fisher Scientific based on the manufacturer’s instructions. The plate was evaluated 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 (Thermo Fisher Scientific). The average intensity of 200 objects (cells) per well was quantified. The ratios were then compared between TNF-α-stimulated, treated, and untreated cells.22 (link)
Cytoplasm to nucleus translocation bioapplication software
Manufactured by Thermo Fisher Scientific
The Cytoplasm to Nucleus Translocation BioApplication software is a tool designed to analyze and quantify the translocation of proteins or molecules from the cytoplasm to the nucleus of cells. It provides automated image analysis capabilities to track and measure this subcellular movement.
Automatically generated - may contain errors
Lab products found in correlation
Nf κb activation kit, by Thermo Fisher Scientific (3 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Cellomics arrayscan hcs reader, by Thermo Fisher Scientific (1 mentions)
Cellomics nf κb activation kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using cytoplasm to nucleus translocation bioapplication software
1
Quantifying NF-κB Activation in TNF-α-Stimulated Cells
2
NF-κB Translocation Assay in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded in 96-well collagen-coated plates at a density of 2 × 104 cells per well. Cells were pretreated with CPZ (20 μM) for 3 h and stimulated with nucleocapsid (1 μg/mL) for 1 h. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. After 3 washes with PBST washing buffer (PBS, 0.1% Tween-20), the cells were stained with NF-κB p65 (D14E12) Alexa Fluor 488 conjugated antibody (Cell Signaling, Danvers, MA, USA) overnight at 4 °C. Then, the cells were washed 3 times with PBST and incubated with nuclear staining solution (2 μg/mL Hoechst 33342 in PBS) for 30 min. Fluorescence was measured using the Cellomics ArrayScan HCS Reader and image analysis software (Thermo Fisher Scientific). The Cytoplasm to Nucleus Translocation BioApplication software (Thermo Fisher Scientific) was used to calculate the difference in nuclear and cytoplasmic fluorescence intensity. Nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) and analyzed subsequently using Western blotting.
3
NF-κB Activation Assay in MCF-7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, MCF-7 cells (1.0 × 104) were seeded into a 96-well plate. On the next day, the cells were treated with a different dosage of A and B for 3h ours and stimulated with 1 ng/mL tumor necrosis factor alpha (TNF-α) for 30 minutes. Medium was then removed and the cells were fixed and stained with a nuclear factor kappa B (NF-κB) activation kit from Thermo Fisher Scientific according to the manufacturer’s instructions. The plate was analyzed by an ArrayScan high content screening reader. Finally, the intensity ratio of cytoplasmic and nuclear NF-κB was determined using Cytoplasm to Nucleus Translocation BioApplication software (Thermo Fisher Scientific). The average intensity of 200 objects (cells) per well was quantified. All values are mean of three experiments. The ratios were compared between TNF-α-stimulated, treated, and untreated cells30 (link)48 .
4
NF-κB Activation Assay for Biseugenol B
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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)
5
NF-κB Activation Assay in HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVECs were seeded overnight in a 96-well plate at 6,000 cells/well. The cells were either pretreated for 1 hour with either the positive control curcumin (6.25 μg/mL), the different concentrations of AA, or were left UT. The cells were stimulated with 2 ng/mL TNF-α for 30 minutes. The medium was discarded and cells were fixed and stained using Cellomics® NF-κB activation kit from Thermo Fisher Scientific, according to the manufacturer’s instructions. The assay plate was evaluated on the ArrayScan high content screening (HCS) Reader. The Cytoplasm to Nucleus Translocation Bio-Application software (Thermo Fisher Scientific) was used to calculate the ratio of cytoplasmic and nuclear NF-κB intensity.26 (link) The ratios were then compared among stimulated, treated, and UT cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!