Interaction of bacterial toxin with leukocytes measured by flow cytometry

Enterobacter cloacae toxin was purified in the form of monomer and polymer. Both forms stimulated the generation of reactive oxygen species (ROS) at sublytic concentration; the oxidative stress produced was studied by using chemiluminescence (CL). The alteration generated caused death of leukocytes, especially at high toxin concentration. Polymeric toxin produced more oxidative stress than the monomeric one. Cytometry allowed the detection of more toxin binding to neutrophils rather than to monocytes or lymphocytes. There was binding at 4 degrees C, and the amount of toxin in the cells increased at 37 degrees C. The interaction of toxin with leukocytes was evident even after 100 degrees C treatment of toxin during 5 min. The incubation with 2-mercaptoethanol was not necessary for toxin binding.