Adherence of S-layer protein-coated cell wall fragments to IPEC-1 cells
CWF to be used as uncoated controls were labeled by EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions by adding 0.5 mg of label per 500 μg of CWF (dry weight). IPEC-1 cells grown on Thincert™ wells were washed once with PBS, and 80 μg of Slp-coated or uncoated CWF in the total volume of 100 μl DMEM/Ham’s F-12 [1:1] medium was added per well, corresponding to approximately 8 μg (0.13-0.18 nmol) of each S-layer protein per well containing 2.5 x 105 IPEC-1 cells. The plate was incubated for two hours at +37°C and 5% CO2 followed by four washes with PBS. The cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes at room temperature and washed three times with 0.1 M sodium phosphate buffer (pH 7.4). Slp-coated CWF were detected by an indirect immunofluorescence staining with Slp-specific immunoglobulins (20 μg/ml, purified by Hi Trap columns, GE Healthcare, Little Chalfont, UK) and AlexaFluor488-conjugated secondary antibodies (2 μg/ml, LifeTechnologies, Carlsbad, California), all in PBS-0.1% BSA, and uncoated cell walls were detected by staining with AlexaFluor488-conjugated streptavidin (2 μg/ml, LifeTechnologies, Carlsbad, California) in PBS-0.1% BSA. The bottoms of the Thincert™ wells were prepared for microscopy and observed in a Leica DM 4000B epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). The mean number of adherent CWF was quantitated from 20 randomly selected fields of 3.5 x 104 μm2, and representative photographs were taken with the Olympus DP70 digital camera system with the cellP imaging software (Olympus Corp., Tokyo, Japan).