Fibronectin culture plate coating

The mechanism by which Cell-Tak protein accomplishes this efficiency at the molecular level is addressed with the observation that different cell types attach with the same kinetics (Figure 2A). The lymphoma line (U-937), BHK cells, and bovine corneal endothelial cells attach with varying efficiencies to plastic (Figure 2B) but exhibit the same rate and overall efficiency on Cell-Tak protein. The attachment lacks the specificity that would indicate receptor-mediated attachment and supports the roll of nonspecific interactions. Moreover, other data (not shown) suggest that the attachment of cells is due to Cell-Tak and not to any component such as fibronectin that is found in serum. This is seen in experiments involving the preincubation of Cell-Tak-coated dishes with either fibronectin or serum. Cell-Tak-coated plates were preincubated with cell culture medium containing 20% calf serum for 30 min, and fibronectin was dried onto other plates coated with Cell-Tak before U-937 cells were plated. Cell attachment on fibronectin-coated Cell-Tak plates was identical to attachment to Cell-Tak alone, and attachment efficiency was inhibited by 40% on plates preincubated with serum. 

Fig. 2.2. Positive and negative adhesion of fibroblasts on fibronectin and tenascin coating of a tissue culture dish, after saturation with BSA. After I h of plating in serum-free medium, cells appear loosely attached on BSA, well spread on fibronectin and not attached on tenascin. Crystal violet staining. (Photograph courtesy of Ruth Chiquet, Friedrich Miescher Institute.)...

Aspirate fibronectin solution and rinse once in RPMI culture medium. Add 250 pi RPMI culture medium until ready to plate cells. Fibronectin-coated slides may be stored up to 2 days at 4°C (Fig. 3b). 

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