NF-kappa B is activated at sites of many chronic inflammatory diseases (atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis...) where it plays a central role in immune response and inflammation. NF-kappa B can induce the transcription of proinflammatory cytokines, chemokines, adhesion molecules, metalloproteinases,...

Here BIOalternatives developed an automated in vitro assay for compounds interfering with NF-Kappa B nuclear translocation, based on immunofluorescence/image analysis, in 96 wells micro-plate format.

Biological model :
Normal human epidermal keratinocytes (other models available).

Culture conditions :
Cells in 96 wells micro-plates, incubated in KSFM at 37°C, 5% CO2.

Treatement and stimulation :
As an example, the conditions presented are:

• Control medium.
• Stimulation with a mix of pro-inflammatory cytokines.
• Stimulation with a mix of pro-inflammatory cytokines + NF-Kappa B inhibitor.
  

Three wells were carried out per experimental condition. After a stimulation time of twenty minutes, cultures were fixed and permeabilized.

Immunofluorescence :
The nuclei of the cells were labeled with Hoechst dye. NF-Kappa B was labeled by an anti-NF-Kappa B monoclonal antibody Alexa Fluor™ 488-conjugated.

Automated analysis :
Images and analysis of NF-Kappa B nuclear translocation were automaticaly carried out using In Cell Analyzer 1000 and In Cell Analyzer 1000 v3.2 software (GE Healthcare). For each condition, 3 images per well (9 images per condition) were taken at 20x magnification. The results were expressed as number of translocation event per image. Statistical analysis was performed with the Student unpaired t-test.

The results are shown on Image 1.

After cytokine mix stimulation (Image 2b), the nuclear translocation of NF-Kappa B was strong compared to the control (Image 1b) in which the NF-Kappa B was cytoplasmic.

The addition of NF-Kappa B inhibitor clearly inhibited the NF-Kappa B nuclear translocation (Image 3b).

Image 1: Effects of the NF-Kappa B inhibitor on NF-Kappa B nuclear translocation after cytokine stimulation (green: NF-Kappa B immunolabelling; blue: nuclei staining by Hoechst dye).

In conclusion, this automated assay using In Cell Analyzer 1000 is useful for the screening of compounds.

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