Fluorescence depolarization as an early measure of T lymphocyte stimulation

J Immunol Methods. 1997 Feb 14;201(1):15-24. doi: 10.1016/s0022-1759(96)00189-5.

Abstract

We have used the Cellscan, an apparatus capable of measuring optical properties of individual cells, to study changes in fluorescence polarization associated with T cell stimulation. We show that the fluorescence polarization of human peripheral blood lymphocytes (PBL) labeled with fluorescein diacetate (FDA) is markedly reduced upon exposure to the mitogenic lectins phytohemagglutinin (PHA), concanavalin A (ConA), or to phorbol esters. Methyl alpha-D-mannopyranoside (alphaMM) is able to reverse the depolarizing effect induced by ConA as long as the cells are not committed to proliferate. H7 and staurosporin, both inhibitors of protein kinase C (PKC), inhibit the depolarization induced by PHA. The mitogen-induced depolarization is dependent on metabolic energy. The results support the use of fluorescence depolarization of FDA-labeled PBL, monitored by the Cellscan, as a sensitive means of measuring early lymphocyte stimulation.

MeSH terms

  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / pharmacology
  • Concanavalin A / pharmacology
  • Cytoplasm / ultrastructure
  • Enzyme Inhibitors / pharmacology
  • Fluoresceins
  • Fluorescence Polarization
  • Humans
  • Lymphocyte Activation*
  • Lymphocyte Count
  • Phosphatidylinositols / physiology
  • Phytohemagglutinins / pharmacology
  • Protein Kinase C / antagonists & inhibitors
  • Staurosporine / pharmacology
  • T-Lymphocytes / physiology*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Time Factors

Substances

  • Enzyme Inhibitors
  • Fluoresceins
  • Phosphatidylinositols
  • Phytohemagglutinins
  • Concanavalin A
  • 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
  • Protein Kinase C
  • Staurosporine
  • Tetradecanoylphorbol Acetate
  • diacetylfluorescein