Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System

Roelse, Margriet; Henquet, Maurice; Verhoeven, H. A.; Ruijter, N.C.A. de; Wehrens, Ron; Lenthe, Marco S van; Witkamp, Renger F.; Hall, Robert D.; Jongsma, Maarten A.

doi:10.3390/s18020602

Cited by 3 publications

(9 citation statements)

References 43 publications

(61 reference statements)

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“…Reverse-transfected cell arrays were prepared and measured as previously described in [6]. The genes encoding bitter receptors were obtained from genomic DNA by PCR amplification and cloned into pcDNA3 containing the N-terminal sstr3 tag (gift from Dr. Wolfgang Meyerhof, German Institute of Human Nutrition Potsdam-Rehbrücke, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Upon stimulus by their respective ligands, receptors will induce a signal transduction leading to an increased calcium ion concentration in the cytosol. This can be monitored in real time by means of FRET (Förster Resonance Energy Transfer) imaging [6], here using a Leica fluorescent stereo microscope (Leica M205FA with DFC 345 FX camera). Two channels were monitored, CFP (ET CFP 10447409, excitation 436/20 and emission 480/40) and YFP (ET FRET 10450566, excitation 436/20 and emission 535/30), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…For example, the human tongue can be emulated on a chip by an array containing G-protein coupled receptors (GPCRs), e.g ., in the form of reconstituted receptor proteins [4] or vesicles [5]. Another possibility is formed by living cells expressing the genes coding for particular GPCRs, produced by either reverse-transfecting a generic cell line on the chip [6] or spotting pre-transfected cells [7]. Such a tongue-on-a-chip allows direct access to the original taste signal, before the signal is further transmitted via neurons, and processed and interpreted by the human brain.…”

Section: Introductionmentioning

confidence: 99%

“…Here, we are focusing on microfluidic receptomics chips containing receptor cell arrays generated by reverse transfection of DNA arrays and with ectopic expression of different GPCRs and a generic calcium-ion sensor protein, Twitch2B [6, 9]. The chips were printed with plasmid DNA encoding a GPCR gene and a calcium sensor gene.…”

Section: Introductionmentioning

confidence: 99%

“…Since the array can contain hundreds of spots, it is possible to accomodate many different receptors and at the same time have a relatively large number of spots for each receptor type, leading to more precise estimates. For each spot on the array, fluorescence time series are measured at two different wavelenghts which are, in a series of steps, transformed in response values for each spot upon exposure to a sample [6]. The goal of the statistical analysis, the main focus of this paper, is to be able to unambiguously and objectively discriminate between samples in terms of receptors affected.…”

Section: Introductionmentioning

confidence: 99%

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Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays

et al. 2019

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Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays

et al. 2019

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Tongue-on-a-Chip: Parallel Recording of Sweet and Bitter Receptor Responses to Sequential Injections of Pure and Mixed Sweeteners

Roelse,

Krasteva,

Pawlizak

et al. 2024

J. Agric. Food Chem.

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A microfluidic tongue-on-a-chip platform has been evaluated relative to the known sensory properties of various sweeteners. Analogous metrics of typical sensory features reported by human panels such as sweet taste thresholds, onset, and lingering, as well as bitter off-flavor and blocking interactions were deduced from the taste receptor activation curves and then compared. To this end, a flow cell containing a receptor cell array bearing the sweet and six bitter taste receptors was transiently exposed to pure and mixed sweetener samples. The sample concentration gradient across time was separately characterized by the injection of fluorescein dye. Subsequently, cellular calcium responses to different doses of advantame, aspartame, saccharine, and sucrose were overlaid with the concentration gradient. Parameters describing the response kinetics compared to the gradient were quantified. Advantame at 15 μM recorded a significantly faster sweetness onset of 5 ± 2 s and a longer lingering time of 39 s relative to sucrose at 100 mM with an onset of 13 ± 2 s and a lingering time of 6 s. Saccharine was shown to activate the bitter receptors TAS2R8, TAS2R31, and TAS2R43, confirming its known off-flavor, whereas addition of cyclamate reduced or blocked this saccharine bitter response. The potential of using this tongue-on-a-chip to bridge the gap with in vitro assays and taste panels is discussed.

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The Effect of Calcium Buffering and Calcium Sensor Type on the Sensitivity of an Array-Based Bitter Receptor Screening Assay

et al. 2019

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Abstract The genetically encoded calcium sensor protein Cameleon YC3.6 has previously been applied for functional G protein–coupled receptor screening using receptor cell arrays. However, different types of sensors are available, with a wide range in [Ca2+] sensitivity, Hill coefficients, calcium binding domains, and fluorophores, which could potentially improve the performance of the assay. Here, we compared the responses of 3 structurally different calcium sensor proteins (Cameleon YC3.6, Nano140, and Twitch2B) simultaneously, on a single chip, at different cytosolic expression levels and in combination with 2 different bitter receptors, TAS2R8 and TAS2R14. Sensor concentrations were modified by varying the amount of calcium sensor DNA that was printed on the DNA arrays prior to reverse transfection. We found that ~2-fold lower concentrations of calcium sensor protein, by transfecting 4 times less sensor-coding DNA, resulted in more sensitive bitter responses. The best results were obtained with Twitch2B, where, relative to YC3.6 at the default DNA concentration, a 4-fold lower DNA concentration increased sensitivity 60-fold and signal strength 5- to 10-fold. Next, we compared the performance of YC3.6 and Twitch2B against an array with 11 different bitter taste receptors. We observed a 2- to 8-fold increase in sensitivity using Twitch2B compared with YC3.6. The bitter receptor arrays contained 300 spots and could be exposed to a series of 18 injections within 1 h resulting in 5400 measurements. These optimized sensor conditions provide a basis for enhancing receptomics calcium assays for receptors with poor Ca2+ signaling and will benefit future high-throughput receptomics experiments.

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Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System

Cited by 3 publications

References 43 publications

Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays

Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays

Tongue-on-a-Chip: Parallel Recording of Sweet and Bitter Receptor Responses to Sequential Injections of Pure and Mixed Sweeteners

The Effect of Calcium Buffering and Calcium Sensor Type on the Sensitivity of an Array-Based Bitter Receptor Screening Assay

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