Towards the miniaturization of GPCR-based live-cell screening assays

Martins, Sofia Aires M.; Trabuco, João R. C.; Monteiro, Gabriel A.; Chu, V.; Conde, J.P.; Prazeres, D.M.F.

doi:10.1016/j.tibtech.2012.07.004

Cited by 31 publications

(25 citation statements)

References 77 publications

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“…A related approach is to combine information on the FRET biosensor readouts with spatial and temporal information on cell shape or cell behavior (van Unen et al, 2015a). The combination of FRET biosensors and microfluidics approaches (Martins et al, 2012;Sackmann et al, 2014) can be used to solve more detailed questions around GPCRs by delivering more precisely defined stimulations to cells (for example, repeated stimuli or gradients).…”

Section: Discussionmentioning

confidence: 99%

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

et al. 2016

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Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H 1 R, H 2 R, H 3 R, and H 4 R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H 1 R activates Gaq. We also observed that H 1 R activates Gai, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gas, we found that the H 2 R induces Gaq-mediated calcium release. The H 3 R and H 4 R activated Gai with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.

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Section: Discussionmentioning

confidence: 99%

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

et al. 2016

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“…Microfluidics and microarray technologies are currently used in this context to monitor cellular signaling in a highly parallelized and automated fashion typically in nano- to picoliter volumes [1], [2]. Downscaling to smaller volumes using intact mammalian cells is not feasible with such approaches.…”

Section: Introductionmentioning

confidence: 99%

Downscaling the Analysis of Complex Transmembrane Signaling Cascades to Closed Attoliter Volumes

et al. 2013

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Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell’s plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

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“…Cell functional assays represent a higher level of validation for scientific hypothesis and development in biological and biomedical research, and can also be used for clinical disease assessment (Albini and Benelli 2007;Hong and Zu 2013;Kepp et al 2011;Liang et al 2007;Martins et al 2012). In addition to chemotaxis assay as described in this study, we envision that the MS 2 technology can be developed to enable a range of cell functional assays such as cell viability assay, apoptosis assays, cell proliferation assay, cell differentiation assay, cell invasion assay, wound healing assays, cell killing assay, live cell signaling assays, and cell-based organ-on-chip imaging and monitoring.…”

Section: Discussionmentioning

confidence: 99%

Mkit: A cell migration assay based on microfluidic device and smartphone

Yang

Peretz‐Soroka

et al. 2018

Biosensors and Bioelectronics

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Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS 2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS 2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS 2 -based cell functional assay for testing cell migration (the M kit ). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the M kit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the M kit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the M kit . In addition to research applications, we demonstrated the effective use of the M kit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed M kit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications.

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Towards the miniaturization of GPCR-based live-cell screening assays

Cited by 31 publications

References 77 publications

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes

Downscaling the Analysis of Complex Transmembrane Signaling Cascades to Closed Attoliter Volumes

Mkit: A cell migration assay based on microfluidic device and smartphone

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