Cell scraper

IEC-6 cells were seeded onto 6-well plates at 5 × 10⁵ cells per well in 3 mL of DMEM containing 10% FBS, 4 µg/mL insulin, 100 U/mL penicillin, and 100 µg/mL streptomycin. After 3 days of culture, HK L-137 (500 μg/mL) was added. After 24 h, the supernatant was removed, and the wells were washed with ice-cold PBS. After removal of PBS, ice-cold RIPA buffer supplemented with protease inhibitor cocktail tablets and phosphatase inhibitor tablets was added, and the cells were collected with a cell scraper (Techno Plastic Products AG, Trasadingen, Switzerland). After 1 h on ice, the extract solution was centrifuged at 16,260g for 30 min at 4 °C, and the supernatant was collected. Total protein levels in the preparations were determined with a Micro Bicinchoninic Acid (BCA) Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA).

CB and GB were developed in the absence and presence of Viz-S for 24 h, as described previously, in a 6-well plate. The supernatant was aspirated and the biofilm was washed twice except for CB in the 50 μM Viz-S group, as the biofilm developed in the presence of 50 μM Viz-S detached readily from the surface by a washing step. The biofilms were collected using a cell scraper (TPP Techno Plastic Products AG, Trasadingen, Switzerland). The collected biofilm was washed twice with PBS. The bacterial suspension was mechanically pulverized with Lysing Matrix B (MP Biomedical, Santa Ana, CA, USA) using a MagNA Lyser (Roche Diagnostic GmbH, Penzberg, Germany) at a speed of 7000 rpm for 30 s. The amount of protein in the biofilms was determined using a bicinchoninic acid (BCA) Protein Assay Kit (T9300A; Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. After incubation for 30 min, absorbance at 550 nm was measured using a colorimeter. The amount of carbohydrate in the biofilm was measured as described previously [12 (link)] with slight modification. Briefly, 500 μl of a 5% aqueous solution of phenol was added to 500 μl of supernatant, incubated for 20 min, and then 2.5 ml of concentrated sulfuric acid was added. The OD at 490 nm was measured using a colorimeter. This assay was performed with a total of five replicates per treatment.