Lymphocyte bacterial rosette test: methodological details and effect of metabolic inhibitors and divalent cations

Lymphocytes bind certain bacteria. This property has been utilized in the lymphocyte bacterial rosette assay to identify T and B cell subsets. Here we determined the optimum conditions for the assay, studied the effect of metabolic inhibitors and divalent cations and compared bacterial rosette-forming lymphocyte subpopulations with those defined with monoclonal antibodies. The strains used were Brucella melitensis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Optimum attachment was obtained at 4 degrees C in 6% BSA with ultrasonicated bacteria. Pretreatment of lymphocytes with the microfilament-disruptive drug cytochalasin B suppressed the binding of bacteria, whereas colchicine (inhibitor of microtubules), puromycin (inhibitor of translation), sodium azide (inhibitor of oxidative phosphorylation) and 2-deoxyglucose (inhibitor of glycolysis) had no effect. Divalent cations were required for the attachment of bacteria. B. melitensis bound to DR-positive cells, whereas the other bacterial strains rosetted OKT4- OKT8- and DR-positive cells without exhibiting helper or suppressor T cell or B lymphocyte specificity.