Flow cytometric detection of membrane potential changes in murine lymphocytes induced by concanavalin A.
The effect of the mitogenic lectin concanavalin A on the membrane potential of murine lymphocytes was investigated by observing the fluorescence of cells stained with carbocyanine and oxonol dyes. We describe a rapid and reliable method for detecting lectin-induced membrane potential changes in individual cells by flow cytometric analysis of oxonol fluorescence. By 10 min after addition of lectin to suspensions of isolated cells from lymph node, 7-15% of the cells have responded by releasing oxonol dye, indicating a membrane hyperpolarization. The dose onset of this response is similar to that for mitogenesis, which was assessed by measuring [3H]thymidine incorporation. The effect is abolished by alpha-methyl mannoside (100mM), which prevents concanavalin A from binding to the cells, but not by fucose (100mM). When cells are treated with lectin in medium from which Ca2+ has been omitted or to which quinine (0.5mM) has been added, a membrane depolarization is observed. Since these are conditions under which activation of plasma membrane Ca2+-dependent K+ channels is prevented, these findings support the view that the early hyperpolarization of these cells is brought about by an increase in intracellular free [Ca2+].
D000818:Animals; D001463:Barbiturates; D002118:Calcium / Q000494:pharmacology; D002232:Carbocyanines; D003208:Concanavalin A / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005643:Fucose / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects*; D008214:Lymphocytes / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D008766:Methylmannosides / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D013858:Thiobarbiturates
D001463:Barbiturates; D002232:Carbocyanines; D008766:Methylmannosides; D013858:Thiobarbiturates; C019029:oxonol dyes (barbiturate); D003208:Concanavalin A; D005643:Fucose; C025284:bis(1,3-diethylthiobarbiturate)trimethineoxonol; C008466:methylmannoside; D002118:Calcium