Bacterial adhesion assay

HT-29 cells, cultured in a 24-multi-well plate at 2 x 10⁵ cells per well at 37°C in an incubator with 5% CO₂ for 1 week until the cells reached confluence, were used for adhesion assay as previously described [24]. Briefly, V. cholerae was grown at 37°C overnight in LB broth and washed three times with PBS. Then, the bacteria (1 x 10⁶ CFU/ml) were incubated with serially-diluted sera or specific isotype-depleted sera for 1 h at 4°C and added to a 24-well plate containing HT-29 cells. After 1 h incubation with gentle shaking, HT-29 cells were washed with PBS to remove unbound bacteria and lysed cells using 0.2% Triton X-100 for 10 min. The lysed cells were diluted and plated on LB agar plate to determine the number of adherent bacteria.