Early changes in concanavalin A-stimulated lymphocytes detected by the fluorescent probe N-phenyl-1-naphthylamine.
The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 micrograms/ml concanavalin A (Con A) for 30 min at 37 degrees C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by 3H-thymidine assay. Increased fluorescence was not observed in the presence of 10 mM alpha-methyl mannoside, 5 mM sodium azide, 10(-5) M cytochalasin B, or Ca2+-free culture medium. However, incubation with 10(-5) M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response. Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweek mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.
D015057:1-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D002412:Cations / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D003208:Concanavalin A / Q000494:pharmacology*; D003571:Cytochalasin B / Q000494:pharmacology; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D005456:Fluorescent Dyes; D037102:Lectins / Q000494:pharmacology; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D013050:Spectrometry, Fluorescence; D013601:T-Lymphocytes / Q000276:immunology*
D002412:Cations; D005456:Fluorescent Dyes; D037102:Lectins; D003208:Concanavalin A; D003571:Cytochalasin B; C005444:N-phenyl-1-naphthylamine; D015057:1-Naphthylamine; D003078:Colchicine