Page 12 - BM 2011 Vol.2 No.4

Bioscience Methods
BM 2011, Vol.2, No.4, 21-30 http://bm.sophiapublisher.com
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medium) which is MEM supplemented with 5%~10%
(v/v) foetal bovine serum (FBS), 1.5 mmol/L L-glutamine
(Invitrogen), 50 µg/mL gentamicin (Invitrogen), 1 µg/mL
of cholera toxin (Sigma-Aldrich, St Louise, MO,
USA), 0.1 µg/mL of murine epidermal growth factor
(Sigma-Aldrich) and 5 µg/mL insulin from bovine
pancreas (Sigma-Aldrich). Monolayers of HCE cells
were grown at 37 in a humidified atmosphere of 5%℃
CO2 in T 25 or T75 tissue culture flasks (Becton-
Dickenson Labware, Oxnard, CA, USA).
3.8 Bacterial adhesion assay
100 µL of 5×106 colony forming units (CFU)/mL
Paer6294-GFP was mixed with 100 µL of serially
diluted mucins (50~200 µg/mL for bell mucin or
62.5~500 µg/mL for exudate mucin). The bacteria
were allowed to bind to mucin at 20 for 2 h with℃
agitation. The mixture (100 µL) was then added to
HCE cells (1×105/well) grown in black 96- well plates
(Greiner Bio-One GmbH, Germany), and allowed to
incubate at 37 for 1 h. BSA at the highest℃
concentration of mucin (100 µg /mL) was used as the
negative control. All assays were performed in
duplicate and the experiment repeated twice. Unbound
bacteria were removed by washing the wells with PBS
(×2) and the GFP fluorescence intensity of each well
was measured (emission 535 nm, excitation 485 nm)
using a Tecan SpectroFluor Plus. The numbers of
adherent Paer6294- GFP in the wells were interpolated
from a standard curve, which was run with each
experiment. Briefly, 100 µL serial dilutions of Paer6294-GFP
(100×105, 50×105, 25×105, 12.5×105, 6.25×105,
3.12×105, 1.56×105 and 0.78×105 CFU/mL) were
transferred in duplicates into a black 96-well plate
containing 1×105/well of HCE cells. The bacteria were
allowed to bind to the cells at 37℃ for 1 h after which
the cells were washed twice with PBS and the
fluorescence data were measured. A standard curve
was created by plotting the mean fluorescence data for
each concentration against bacterial concentration.
3.9 Monosasccharide inhibition
The monosaccharides used in this assay represented
those major monosaccharides present on bell mucin as
determined by the compositional analysis described
above. They were tested in a bacterial anti-adhesion
assay. D (+) galactose (Gal), N-acetylgalactosamine
(GalNAc), and D (+) glucose (Glc) (Sigma-Aldrich)
were prepared as 50 mmol/L, 25 m mol/L and
12.5 mmol/L solutions in PBS. 200 μL of each
mono- saccharide solution was incubated with 200 μL
of Paer6294 -GFP (1×108 CFU/mL) in MEM in the
wells of a 24-well plate and then incubated for 1 h at
20℃with gentle shaking. Following this incubation,
100 μL of the Paer6294 -GFP and monosaccharide
mixture was applied to HCE cells (seeded at 2.5×
105/well) and incubated for 1 h (37℃ , 5% CO2) in a
96-well plate. Following 3 washes with MEM,
fluorescence intensities in wells were measured and
the number of adherent bacteria was calculated from a
standard curve, constructed as described above.
Authors’ contributions
RP carried out the mucin extraction and purification,
participated in compositional analyses, and contributed to the
writing, RT participated in the design of the study and edited
the manuscript, RA performed the cytotoxicity test. BX and ZZ
performed bacterial adherence assays and analysed the data.
MW coordinated the project and helped to edit the manuscript.
KK conceived the study, and participated in the experimental
design and coordination and wrote the manuscript. All authors
read, and approved the final manuscript.
Acknowledgements
We thank Gene Wijffels and Michelle Colgrave for the review
of the manuscript.
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