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All-Optical Miniaturized Co-culture Assay of Voltage-Gated Ca2+ Channels

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2173)

Abstract

Light-activated proteins enable the reversible and spatiotemporal control of cellular events in optogenetics. Optogenetics is also rapidly expanding into the field of drug discovery where it provides cost-effective and noninvasive approaches for cell manipulation in high-throughput screens. Here, we present a prototypical cell-based assay that applies Channelrhodopsin2 (ChR2) to recapitulate physiological membrane potential changes and test for voltage-gated ion channel (VGIC) blockade. ChR2 and the voltage-gated Ca2+ channel 1.2 (CaV1.2) are expressed in individual HEK293 cell lines that are then co-cultured for formation of gap junctions and an electrical syncytium. This co-culture allows identification of blockers using parallel fluorescence plate readers in the 384-well plate format in an all-optical mode of operation. The assay is transferable to other VGICs by modularly combining new and existing cell lines and potentially also to other drug targets.

Key words

Optogenetics High-throughput screening 384-well plate Voltage-gated ion channel Syncytium CaV1.2 Channelrhodopsin All-optical FLIPR 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Cell BiologyAXXAM S.p.AMilanItaly
  2. 2.Australian Regenerative Medicine Institute (ARMI), Faculty of Medicine, Nursing and Health SciencesMonash UniversityClaytonAustralia
  3. 3.European Molecular Biology Laboratory Australia (EMBL Australia)Monash UniversityClaytonAustralia

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