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Cellomics nf κb activation kit

Manufactured by Thermo Fisher Scientific
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The Cellomics NF-κB activation kit is a laboratory equipment product designed to measure and analyze the activation of the NF-κB transcription factor in cells. It provides a quantitative, high-throughput method to assess NF-κB activity in response to various stimuli.

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5 protocols using cellomics nf κb activation kit

1

Girinimbine Inhibits LPS-Induced NF-κB Activation

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RAW 264.7 cells were cultured in 96-well plates at a density of 5×103 cells/well and pretreated with girinimbine for 24 hours. Following the pretreatment, RAW 264.7 cells were stimulated with LPS (10 ng/mL) for 30 minutes. Control wells received similar treatment conditions expect for girinimbine pretreatment. The cells were then stained using a Cellomics NF-κB activation kit (Thermo Fisher Scientific) according to the kit instructions. RAW 264.7 plates were evaluated on the ArrayScan HCS Reader (Cellomics; Thermo Fisher Scientific). Ratios of cytoplasmic and nuclear factor-kappa B (NF-κB) intensity29 (link) (average intensity of 200 cells/well) were calculated by Cytoplasm to Nucleus Translocation BioAp-plication software (Cellomics, Inc, Pittsburgh, PA, USA).
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2

NFkB Translocation Assay in Macrophages

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Murine (RAW264.7) macrophages were cultured in 4-well chamber slides (Chamber slide system, Nunc®, Denmark) at 4 x 105 cells/chamber in triplicate. Pre-treatment and incubation with antigens/mycobacteria (Table 1) was carried out for 30 minutes (based on a series of optimization experiments to determine the optimum minimum time of incubation to generate appreciable NFκB cytoplasmic to nuclear translocation). Immunofluorescence staining of the cells was carried out using the Cellomics® NFκB activation kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. Briefly, cells were fixed with 4% paraformaldehyde, permeablized and incubated with rabbit anti-NFκB polyclonal primary antibody for 1 h. The cells were then incubated with DyLight488 goat anti-rabbit secondary antibody and Hoechst dye. Visualization was by an Olympus® BX61 fluorescence microscope. Image analysis was by ImageProPlus v 5.0 software (Media Cybernetics). The nuclear to cytoplasmic fluorescence intensity was calculated by dividing nuclear fluorescence by cytoplasmic fluorescence. The entire process was repeated for 6 images (two sets for each replicate chamber). The average nucleus: cytoplasm (N: C) NFκB fluorescence was then calculated for each treatment group and data plotted as a histogram.
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3

NF-κB Inhibition by LG and LGC

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The inhibitory activity of NF-κB through LG and LGC was analyzed using the CellomicsArray Scan HCS system. This assay was performed using the Cellomics NF-κB activation kit (Thermo Fisher Scientific, USA).20 After treatment of the SKOV-3 cancer cells with LG and LGC (10 µg.mL−1, 24 h), TNF-α (10 ng.mL−1, 30 min) was used for stimulation. After the treatment period, fixation and staining were performed according to the kit protocol. The Array Scan HCS Reader and Cytoplasm to Nucleus Translocation Bioapplication software were used for data analysis.
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4

Quantifying NF-κB Nuclear Translocation

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Cells were grown in black 96-well glass bottom plates (Costar). Measurement of NF-κB translocation from the cytoplasm to the nucleus was performed as previously described [13] (link) using a Cellomics NF-κB Activation Kit (Thermo Fisher Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol. The nucleus was immunostained with Hoechst 33342, and NF-κB was detected with an NF-κB primary antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody. Microscopic images were captured with the MetaFluor Imaging System as described above. Image analysis was performed using NIH ImageJ software (v.3.91, http://rsbweb.nih.gov/ij/) as described by Noursadeghi et al. [21] (link). NF-κB nuclear translocation was determined as an increase in the nucleus∶cytoplasm ratio.
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5

NF-κB Activation Assay in HUVECs

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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.
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