HTRF Human/Mouse Fibronectin Detection Kit HTRF®

Human liver hepatocellular carcinoma cells (HepG2) were plated in a culture-treated 96-well plate (100,000 cells/well) in complete culture medium and incubated overnight at 37°C - 5% CO2. Cells were then incubated for 4 hours with a Protein Transport Inhibitor (1X) or not stimulated (NS). After supernatant collection, cells were lysed with 50 µL of lysis buffer #3 (1X) for 30 minutes at RT under gentle shaking.

16 µL of lysate or diluted supernatant to remain in the linear range of the assay were transferred into a low volume white plate before adding 4 µL of the HTRF Fibronectin detection antibodies. The HTRF signal was recorded after an overnight incubation.

As expected, the Protein Transport Inhibitor blocks the release of Fibronectin in supernatant and increase Fibronectin in cell content. The pharmacological effect is less important in supernatant due to accumulation of secreted soluble Fibronectin.

Primary human lung fibroblasts (HLFs)  were plated in a culture-treated 96-well plate (12,500 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, the cells were treated with increasing concentrations of TGF-ß1 for 48 hours in serum-free culture medium supplemented with 1% BSA. After supernatant collection, the cells were lysed with 50 µL of lysis buffer #3 for 30 minutes at RT under gentle shaking.

16 µL of lysate or supernatant diluted in lysis buffer #3 to remain in the linear range of the assay were transferred into a low volume white microplate before the addition of 4 µL of the HTRF Fibronectin detection antibodies. The HTRF signal was recorded after an overnight incubation. TGF-ß1 treatment results in a dose dependent increase of Fibronectin expression level, which demonstrates the differentiation of fibroblasts into myofibroblasts.

The adenocarcinomic human alveolar basal epithelial cells, A549, was plated in a culture-treated 96-well plate (50,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, the cells were treated with 10 ng/ml of TGF-ß1 for 48 hours in serum-free culture medium supplemented with 1% BSA. The supernatant was collected, and cells were lysed with 50 µL of lysis buffer #3. 16 µL of lysate or supernatant were transferred into a low volume white microplate before the addition of 4 µL of the HTRF Fibronectin detection antibodies. The HTRF signal was recorded after an overnight incubation.

TGF-ß1 treatment results in an increase of Fibronectin expression level in cell lysate and an accumulation of higher quantity in supernatant after 48h treatment.

The mouse fibroblast cell line NIH/3T3 was seeded in a T175 flask, and incubated for 3 days at 37°C. After supernatant collection, the cells were lysed with 3 mL of lysis buffer #3 (1X) for 30 minutes at RT under gentle shaking. For each sample (supernatant or cell lysate), several dilutions in the kit diluent lysis buffer  #3, were assessed to determine the most appropriate conditions to remain in the linear range of the assay. 16 µL of each sample were transferred into a low volume white microplate before the addition of 4 µL of the HTRF Fibronectin detection reagents. 

Results show a nice detection of mouse Fibronectin with high concentration in cell content and supernatant. As expected, concentration in supernatant (around 30 000 ng/ml) is greater than that cell lysate (around 10 000 ng/ml) because there is an accumulation of secretion of Fibronectin. These results show a cross reactivity with mouse model.

Fibronectin is a large glycoprotein of the extracellular matrix that exists in two forms: insoluble (cellular) and as a soluble plasma protein. The primary functions of fibronectin are its involvement in cell adhesion, growth, migration, and differentiation as it bridges the gap between cells and the extracellular matrix (ECM). It is typically secreted by fibroblasts as part of their ECM elaboration role, and is critical to morphogenesis, tissue architecture, and wound repair.

Fibronectin is known to contribute to the wound healing process, as it will accumulate at the site of an injury to help form blood clots and aid in the development of new tissues. Fibronectin levels in serum and plasma have been identified as increasing significantly after major traumas. It is regulated by inflammatory factors and growth factors such as cytokines and TGF-b1, as well as by integrin-dependent signaling. Further, significantly altered Fibronectin levels have been observed in the disease states of certain types of cancer, fibrotic disorders, and also diabetes mellitus.