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Nat Protoc. 2019 Jul;14(7):2205-2228. doi: 10.1038/s41596-019-0178-y. Epub 2019 Jun 24.

High-throughput multicolor optogenetics in microwell plates.

Bugaj LJ1,2, Lim WA3,4,5,6.

Author information

1
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA. bugaj@seas.upenn.edu.
2
Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA. bugaj@seas.upenn.edu.
3
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
4
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
5
Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.
6
Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA, USA.

Abstract

Optogenetic probes can be powerful tools for dissecting complexity in cell biology, but there is a lack of instrumentation to exploit their potential for automated, high-information-content experiments. This protocol describes the construction and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments that allows simultaneous manipulation of common red- and blue-light-sensitive optogenetic probes. The optoPlate-96 enables illumination of individual wells in 96-well microwell plates or in groups of wells in 384-well plates. Its design ensures that there will be no cross-illumination between microwells in 96-well plates, and an active cooling system minimizes sample heating during light-intensive experiments. This protocol details the steps to assemble, test, and use the optoPlate-96. The device can be fully assembled without specialized equipment beyond a 3D printer and a laser cutter, starting from open-source design files and commercially available components. We then describe how to perform a typical optogenetics experiment using the optoPlate-96 to stimulate adherent mammalian cells. Although optoPlate-96 experiments are compatible with any plate-based readout, we describe analysis using quantitative single-cell immunofluorescence. This workflow thus allows complex optogenetics experiments (independent control of stimulation colors, intensity, dynamics, and time points) with high-dimensional outputs at single-cell resolution. Starting from 3D-printed and laser-cut components, assembly and testing of the optoPlate-96 can be accomplished in 3-4 h, at a cost of ~$600. A full optoPlate-96 experiment with immunofluorescence analysis can be performed within ~24 h, but this estimate is variable depending on the cell type and experimental parameters.

PMID:
31235951
DOI:
10.1038/s41596-019-0178-y
[Indexed for MEDLINE]

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