Online citations, reference lists, and bibliographies.
Please confirm you are human
(Sign Up for free to never see this)
← Back to Search

High-throughput Multicolor Optogenetics In Microwell Plates

L. J. Bugaj, W. Lim
Published 2019 · Computer Science, Medicine

Save to my Library
Download PDF
Analyze on Scholarcy
Share
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. A protocol for the assembly and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments in microwell plates. With the provided design files, users can assemble the optoPlate-96 from 3D-printed and laser-cut components.
This paper references
10.1021/sb500305v
Orthogonal optogenetic triple-gene control in Mammalian cells.
K. Müller (2014)
10.1101/548255
Optogenetic control reveals differential promoter interpretation of transcription factor nuclear translocation dynamics
S. Chen (2019)
10.1021/acssynbio.6b00333
A Phytochrome-Derived Photoswitch for Intracellular Transport
M. Adrian (2017)
Optogenetic Control of Ca lcium Oscillation Waveform Defines NFAT as an Integrator of Calcium Load Graphical
Pimkhuan Hannanta-anan (2016)
10.1146/annurev-chembioeng-060816-101254
At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior.
Nicole A. Repina (2017)
10.1021/BI000585+
Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.
M. Salomon (2000)
10.1039/C2LC40478H
Optical microplates for high-throughput screening of photosynthesis in lipid-producing algae.
Meng Chen (2012)
10.1126/science.aar7042
Multiplexed protein maps link subcellular organization to cellular states
G. Gut (2018)
10.1101/055053
An open-hardware platform for optogenetics and photobiology
K. Gerhardt (2016)
10.1073/pnas.1417910112
Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins
Gurkan Guntas (2014)
10.1002/cbic.201800030
Optogenetic Control by Pulsed Illumination
Julia Hennemann (2018)
10.1074/jbc.R100006200
The Phytochromes, a Family of Red/Far-red Absorbing Photoreceptors*
C. Fankhauser (2001)
10.1126/science.1131163
Rapid Chemically Induced Changes of PtdIns(4,5)P2 Gate KCNQ Ion Channels
Byung-Chang Suh (2006)
10.1126/science.aao3048
Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras-Erk pathway
L. J. Bugaj (2018)
10.1039/c4pp00361f
Upgrading a microplate reader for photobiology and all-optical experiments.
Florian Richter (2015)
10.1038/nature08446
Spatiotemporal Control of Cell Signalling Using A Light-Switchable Protein Interaction
Anselm Levskaya (2009)
10.1186/1471-2105-9-482
CellProfiler Analyst: data exploration and analysis software for complex image-based screens
T. Jones (2008)
10.1038/nmeth.1700
Light-based feedback for controlling intracellular signaling dynamics
J. Toettcher (2011)
10.1016/j.jmb.2013.07.036
Programming microbes using pulse width modulation of optical signals.
E. A. Davidson (2013)
10.1186/gb-2006-7-10-r100
CellProfiler: image analysis software for identifying and quantifying cell phenotypes
A. Carpenter (2006)
10.1007/978-1-4939-7154-1_1
Optogenetic Control of Ras/Erk Signaling Using the Phy-PIF System.
Alexander G. Goglia (2017)
10.1038/ncomms9390
Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method
Jia-Ren Lin (2015)



This paper is referenced by
Semantic Scholar Logo Some data provided by SemanticScholar