Flow Cytometry Enables a High-Throughput Homogeneous Fluorescent Antibody-Binding Assay for Cytotoxic T Cell Lytic Granule Exocytosis

Abstract

We developed a homogeneous phenotypic fluorescence end-point assay for cytotoxic T lymphocyte lytic granule exocytosis. This flow cytometric assay measures binding of an antibody to a luminal epitope of a lysosomal membrane protein (LAMP-1) that is exposed by exocytosis to the extracellular solution. Washing to remove unbound antibody is not required. Confirming the assay’s ability to detect novel active compounds, we screened at a concentration of 50 µM a synthetic diversity library of 91 compounds in a 96-well plate format, identifying 17 compounds that blocked by 90% or more. The actions of six structurally related tetracyano-hexahydroisoindole compounds that inhibited by ~90% at a concentration of 10 µM were investigated further. Four reduced elevations in intracellular Ca2+; it is likely that depolarization of the cells’ membrane potential underlies the effect for at least two of the compounds. Another compound was found to be a potent inhibitor of the activation of the mitogen-activated protein (MAP) kinase ERK. Finally, we transferred the assay to a 384-well format and screened the Prestwick Compound Library using high-throughput flow cytometry. Our results indicate that our assay will likely be a useful means of screening libraries for novel compounds with important biological activities.