The wells of Maxisorb plates (Nunc, Roskilde, Denmark) were coated with type I collagen (from rat tail, BD Bioscience, New Jersey, United States) (100 μg/ml) or human fibronectin (Serva, Heidelberg, Germany) (100 μg/ml) for 16 h at 4°C. The collagen-binding ability of the selected strains was tested according to Miljkovic et al. (2015 (link)), while their ability to bind fibronectin was assayed as previously described by Ahmed et al. (2001 (link)). After immobilization, the wells were washed with PBS and blocked with 2% BSA in PBS. When the BSA solution was removed, the wells were washed with PBS and the test cultures (100 μl, 108 CFU/ml) were added. Plates were incubated on an orbital platform shaker for 3 h at 37°C. Then, nonadherent cells were removed by washing the wells three times with 200 μl of PBS. The adherent cells were fixed at 60°C for 20 min and stained with crystal violet (100 μl/well, 0.1% solution) for 45 min. Wells were subsequently washed three times with PBS to remove the excess stain. The stain bound to the cells was dissolved in 100 μl of citrate buffer (pH 4.3) and the absorbance was measured at 595 nm after 45 min using the microtiter plate reader. Results were expressed as the mean of six replicates relative to those of the same non-coated wells. Laboratory strain Lactococcus lactis BGKP1 was used as a positive control.
Human fibronectin
Manufactured by Serva Electrophoresis
Sourced in United States, Germany
Human fibronectin is a high molecular weight glycoprotein found in the extracellular matrix and plasma. It plays a crucial role in cell attachment, cell migration, and wound healing.
Automatically generated - may contain errors
3 protocols using human fibronectin
1
Bacterial Adhesion to Extracellular Matrix
2
Collagen and Fibronectin Binding Assay
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The wells of Maxisorb plates (Nunc, Roskilde, Denmark) were coated with type I collagen (from rat tail, BD Bioscience, New Jersey, United States) (100 μg/ml) or human fibronectin (Serva, Heidelberg, Germany) (100 μg/ml) for 16 h at 4 °C. The collagen-binding ability of the selected strains was tested according to Miljkovic and coauthors [15 (link)] while the ability of tested strains to bind to fibronectin was assayed as previously described by Ahmed and coauthors [16 (link)]. Average of six absorbance values per each strain for collagen- and fibronectin-binding was compared with those of the non-coated wells. Escherichia coli DH5α was used as a negative control, while laboratory strain Lactococcus lactis BGKP1 was used as a positive control.
3
Adhesion of Staphylococci to Fibronectin
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Adhesion of tested bacterial strains to human fibronectin was evaluated according to Peacock et al. (2000) (link). All tested strains of staphylococci were preincubated for 24 h and 48 h in the presence of subinhibitory concentrations of chalcones in 24 well plates as previously described in the section of production of glycocalyx. Each isolate had its own positive control (bacteria cultivated solely in medium at the same laboratory conditions).
Polysorp microtiter plates (NUNC, Denmark) were coated for 1 h at 37 °C with 25 μg/mL of human fibronectin (Serva, Germany). After overnight blockage of the remaining sites at 4 °C with 200 μL of 2% bovine serum albumine (Sigma-Aldrich) in PBS, plates were washed three times with PBS and inoculum of 1×108 bacteria in PBS/well was added in triplicates to each well. Plates were incubated 1 h at 37 °C, rinsed three times with PBS, fixed with 2% glutaraldehyde (TCI Europe) in PBS for 1 h and stained with crystal violet for 5 min. After rinsing with water and air drying, the absorbance was measured at OD405. Two triplicates of PBS without bacteria served as a negative control for each plate. After calculation of the results, absorbance values were expressed as the percentage of the absorbance of the positive control. Each assay was repeated three times.
Polysorp microtiter plates (NUNC, Denmark) were coated for 1 h at 37 °C with 25 μg/mL of human fibronectin (Serva, Germany). After overnight blockage of the remaining sites at 4 °C with 200 μL of 2% bovine serum albumine (Sigma-Aldrich) in PBS, plates were washed three times with PBS and inoculum of 1×108 bacteria in PBS/well was added in triplicates to each well. Plates were incubated 1 h at 37 °C, rinsed three times with PBS, fixed with 2% glutaraldehyde (TCI Europe) in PBS for 1 h and stained with crystal violet for 5 min. After rinsing with water and air drying, the absorbance was measured at OD405. Two triplicates of PBS without bacteria served as a negative control for each plate. After calculation of the results, absorbance values were expressed as the percentage of the absorbance of the positive control. Each assay was repeated three times.
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