FITC-Labeling of Bacteria and Flow Cytometry

Bacteria were washed twice and incubated with 0.1 mg/ml FITC (Sigma-Aldrich) in PBS at room temperature in the dark for 30 minutes. Subsequently, bacteria were washed three times in PBS to remove unbound FITC. For flow cytometry, 721.221 cells were used as carrier cells to facilitate gating. To this end, bacteria were divided into 96-well plates and incubated with 721.221 cells for 30 minutes on ice to allow for bacterial adhesion to the cells (60 million bacteria were placed together with 100,000 721.221 cells per well). Next, cells were washed and incubated with the indicated amount of CEACAM fusion proteins for 1 hour on ice followed by a 30-minute incubation with Alexa Fluor 647-conjugated donkey anti-human IgG (Jackson Immunoresearch). Histograms of cell-bound bacteria stained with CEACAM fusion proteins were gated on FITC-positive cells.

For blocking experiments, 2 µg of CEACAM1-Ig were preincubated with 1 µg of the respective antibodies for 1 hour on ice. Values obtained without blocking antibodies were arbitrarily set to 1 and all other values were normalized accordingly.