Fluorescence activated cell sorter lysing solution

For fluorescent labeling, bacteria were grown in 5 ml LB, washed twice with 1× PBS followed by resuspension in 5 ml 0.1 N sodium carbonate (Sigma) containing 0.5 mg ml⁻¹ fluorescein isothiocyanate (FITC; Sigma) for 1 h at room temperature and protected from light. Bacteria were washed extensively in PBS (three times), resuspended in RPMI/HSA to an OD₆₀₀ of 1.0, and re-diluted five times in RPMI/HSA. After the labeling reaction, 20 μl FITC-labeled WT or ΔCbpD were incubated for 20 min at 37 °C with freshly isolated human blood (160 µl) containing hirudin to prevent coagulation. When indicated, rCbpD_PA at a final concentration of 20 μg ml⁻¹ was added to the samples. The phagocytosis reaction was stopped using a fluorescence-activated cell sorter lysing solution (BD Biosciences). The samples were washed twice with RPMI/HSA, resuspended in the same solution supplemented with 4% paraformaldehyde (PFA, Alfa Aesar), and analyzed by a flow cytometer using CellStream (Luminex). Gating of cells was carried out on the basis of forward and side scatter. The fluorescence intensity (FL) of 10000 gated neutrophils was measured for each sample, and phagocytosis was identified when neutrophils expressed fluorescence. The geometric mean of the fluorescence intensity (GMFI) was calculated using CellStream software.