title: 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 Ca(2+); 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.
authors.affiliation: University of Connecticut at Storrs, Storrs, CT 06269-3125, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Florian
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Lepensky
authors.forename: Ohyun
authors.initials: O
authors.lastname: Kwon
authors.forename: Mark K
authors.initials: MK
authors.lastname: Haynes
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
authors.forename: Adam
authors.initials: A
authors.lastname: Zweifach
mesh_terms: D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D005089:Exocytosis / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007211:Indoles / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D054852:Small Molecule Libraries / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000187:drug effects / Q000276:immunology
chemical_list: D007211:Indoles; D051907:Lysosomal Membrane Proteins; D054852:Small Molecule Libraries