Fluorescent bacterial rosetting of lymphocyte subpopulations. I. Methodology

Fluorochrome-labelled bacteria were tested for their rosette-forming properties with human lymphocytes in suspension. Acridine orange stained human buffy coat cells or isolated mononuclear cells are rosetted with tetramethyl-rhodamine-isothiocyanate (TRITC)-labelled bacterial strains alone (mono-bacterial rosetting test) or simultaneously with a TRITC-labelled strain and a mutant or taxonomically different strain, labelled with fluorescein-isothiocyanate (double bacterial rosetting test [D-BRT]). Suspensions are centrifuged, washed, finally counterstained with ethidium bromide and examined by fluorescence microscopy. The bacterial binding properties of B cells may be studied by using anti-Ig pretreated mononuclear cells and TRITC bacteria (anti-Ig/BRT). In this study the methodology for bacterial staining, mono-, double- and anti-Ig/BRT is given. Estimation of rosette-forming cells is very accurate, easy and quick due to the bright fluorescence of the bacterial 'beads'. Furthermore, broad applicability of the bacterial rosetting phenomenon to study lymphocyte heterogeneity is gained with the fluorescent assay system.