Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

SLAS Discovery
Xiuyi Alexander Yang, Adam Zweifach

Abstract

Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z' > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI's Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody-six actually stimulated PKC activity. Cytometry, which produces higher Z' for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol e...Continue Reading

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Citations

May 10, 2019·International Journal of Molecular Sciences·Jin-Young LeeSeung-Goo Lee
Aug 5, 2020·SLAS Discovery·Haim Bar, Adam Zweifach
Feb 6, 2021·International Journal of Molecular Sciences·Thien-Kim LeSoo-Jin Yeom

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Methods Mentioned

BETA
flow cytometry
FRET
fluorescence-activated cell sorting
flow
FACS
Light Microscopy
PMA

Software Mentioned

Spectramax
FlowJo
GraphPad
Prism

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