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

Author:

Florian Amy E.1,Lepensky Christopher K.1,Kwon Ohyun2,Haynes Mark K.3,Sklar Larry A.3,Zweifach Adam1

Affiliation:

1. University of Connecticut at Storrs, Storrs, CT, USA

2. University of California Los Angeles, Los Angeles, CA, USA

3. University of New Mexico Center for Molecular Discovery, Albuquerque, NM, USA

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.

Publisher

Elsevier BV

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