Cell Signaling
Cell signaling enables cells to sense their environment and rapidly adjust their behavior through biochemical changes such as phosphorylation or shifts in gene expression. These signals are typically initiated at the cell membrane, where receptors detect external cues and trigger downstream pathways.
The xCELLigence Real-Time Cell Analysis (RTCA) System provides a highly sensitive, label-free way to monitor these signaling events. By measuring impedance changes caused by cell attachment, morphology, and adhesion dynamics, xCELLigence detects rapid responses—within minutes of GPCR ligand addition—as well as longer-term effects such as proliferation changes after growth factor stimulation.
Why Use xCELLigence for Cell Signaling Studies?
Real-time, label-free monitoring under physiological conditions.
Sensitive to changes in cell number, size, morphology, and adhesion.
Able to capture both immediate signaling events and long-term functional responses.
Simple workflow with high throughput capability.
Membrane Receptor Signaling
Membrane receptor signaling. Extracellular stimuli are initially “sensed” predominantly through membrane receptors. Transmission of the signal inside the cell can result in regulation via biochemical modifications and or changes to transcription/translation.
Straightforward Workflow
Step1: Seed cells on E-plate
Step2: Adherent cells grow on E-Plate biosensors, enabling continuous, noninvasive measurement.
Step 3: Cells are treated with GPCR agonist/antagonist, RTK ligand/inhibitor, or Nuclear hormone agonist/antagonist.
Step 4: The xCELLigence system collects data automatically inside the CO₂ incubator, tracking dynamic cellular responses as they occur.
GPCR-Mediated Signaling
G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors and are major therapeutic targets. When activated by diverse stimuli, GPCRs trigger downstream pathways that alter cell morphology, adhesion, and proliferation. These changes can precisely be detected by xCELLigence RTCA through its sensitive, label-free impedance technology.
Unlike traditional GPCR assays that rely on engineered reporter systems or forced pathway coupling, xCELLigence measures the integrated cellular response directly in models expressing endogenous receptors, delivering biologically relevant results. This real-time approach captures rapid signaling events as well as longer-term functional outcomes, improving hit quality in screening and enabling deeper insight into receptor pharmacology.
Key Benefits of xCELLigence for GPCR Studies
Flexible: Detects activation across diverse GPCR families.
Broad coverage: Measures signaling through Gs, Gq, Gi, and G12/13 pathways.
Unbiased response: Captures functional selectivity and whole-cell effects.
Physiologically relevant: Works with primary cells, stem cells, and disease-relevant models.
Gs-coupled GPCRs:
Stimulation of Gs-protein-coupled receptor by calcitonin in CHO-K1 cells.
CHO-K1 cells stimulated with calcitonin show rapid, dose-dependent impedance changes, enabling EC₅₀ determination.
CHO-K1 cells endogenously express the calcitonin 1a receptor (C1a). In this study, Cells were starved for 3 hr in 0.5% FCS), then stimulated with Calcitonin ranging from 5 pM to 5 µm. The left panel shows the dynamic response of CHO-K1 cells to Calcitonin stimulation. The calcitonin EC50 was calculated from the dose-response curve with RTCA software (right panel)
Gi-coupled GPCRs:
Stimulation of Gi-protein-coupled receptor by serotonin in CHO-K1 cells.
Endogenous serotonin receptor activation in CHO-K1 cells produces distinguishable kinetic signatures and accurate EC₅₀ values.
CHO-K1 cells endogenously express the serotonin receptor. In this study, Cells were starved for 3 hr in 0.5% FCS), then stimulated with serotonin ranging from 1 pM to 100 um. The left panel show the dynamic response of CHO-K1 cells to serotonin stimulation. The serotoninSerotonin EC50 was calculated from the dose-response curve with RTCA Software (right panel)
Nuclear Hormone-Mediated Signaling
Nuclear Hormone Receptors (NHRs) are intracellular transcription factors that regulate gene expression and play essential roles in endocrine function. Because they remain in their native biochemical environment, cell-based NHR assays provide highly physiologically relevant insights into estrogen, androgen, thyroid hormone, and other signaling pathways - key targets of endocrine-disrupting chemicals (EDCs).
Traditional NHR assays rely on endpoint measurements, missing the dynamic nature of hormone-driven responses. The xCELLigence RTCA system overcomes this limitation by enabling continuous, label-free, noninvasive monitoring of cellular responses, delivering richer, more predictive data with a simple, automated workflow.
Key Benefits of Using xCELLigence for NRH Studies
Label-free nature allows for sensitive detection of endogenous receptor activity.
Kinetic response profiles may be diagnostic for specific pathways.
Ability to differentiate cytotoxicity and proliferation with a single experiment.
Real-time data that reveal optimal assay readout windows.
Time-dependent Cellular Responses to Agonist of Thyroid Receptor
Rat pituitary GH3 cells, which endogenously express thyroid hormone receptors (TR), respond sensitively to T3 stimulation. GH3 cells were seeded on Agilent E-Plates, cultured overnight, and then treated with T3. Cell Index (CI) was continuously monitored for 96 hours and normalized to the time of treatment (arrow). Untreated cells served as a negative control.
Time-dependent Cellular Responses to Agonist of Estrogen Receptor
Rat pituitary GH3 cells also responded robustly to the estrogen receptor (ER) agonist 17β-estradiol (E2). After overnight culture on Agilent E-Plates, cells were treated with E2, and Cell Index (CI) was monitored continuously for 96 hours and normalized to the treatment time (arrow). Untreated cells served as a negative control. The E2-induced increase in proliferation aligns with previously reported ER signaling behavior in GH3 cells.
Receptor Tyrosine Kinase (RTK) Signaling
Receptor tyrosine kinases drive many of the body’s most critical cellular processes—and when their signaling goes wrong, the consequences can include cancer, inflammation, metabolic disease, and more. Understanding RTK activity is essential, but traditional assay platforms are often slow, complex, and prone to interference.
The xCELLigence RTCA system transforms RTK research with a fast, label-free, and truly cell-based approach. By tracking real-time morphological changes triggered by growth factor signaling, xCELLigence reveals dynamic RTK activity with unmatched simplicity and sensitivity.
Why Researchers Choose xCELLigence for RTK Studies
Real-time insights into rapid signaling and long-term cellular effects.
Label-free, noninvasive detection - no dyes, reporters, or assay interference.
Endogenous receptor activity measured directly in physiologically relevant cells.
Streamlined workflows that reduce hands-on time and eliminate assay complexity.
Endogenous Receptor Tyrosine Kinase Short-Term Response
Cells expressing recombinant PDGFRβ were seeded on an E-Plate 96 and grown overnight before serum starvation. They were then exposed to increasing doses of the PDGF inhibitor Imatinib, followed by stimulation with PDGF-BB. Using xCELLigence real-time monitoring, Imatinib produced a clear dose-dependent inhibition of PDGF-induced morphological changes, yielding an IC₅₀ of 135 nM.
Endogenous Receptor Tyrosine Kinase Expressing Cell Line Response to Inhibitor
Cells expressing constitutively active cMET were seeded on an E-Plate 384 and treated with graded doses of the cMET inhibitor Crizotinib. Real-time monitoring with the xCELLigence system revealed a clear, dose-dependent reversal of the cMET-driven morphological signal, producing an IC₅₀ of 87.5 nM.
Literature
GPCR-Mediated Signaling
- Differential Role of Serines and Threonines in Intracellular Loop 3 and C-Terminal Tail of the Histamine H4 Receptor in β-Arrestin and G Protein-Coupled Receptor Kinase Interaction, Internalization, and Signaling. Verweij EWE, Al Araaj B, Prabhata WR, Prihandoko R, Nijmeijer S, Tobin AB, Leurs R, Vischer HF.ACS Pharmacol Transl Sci. 2020 Mar 16;3(2):321-333.
- Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1. Vlachodimou A, IJzerman AP, Heitman LH.Sci Rep. 2019 Sep 24;9(1):13802.
- Label-free impedance-based whole cell assay to study GPCR pharmacology. Doornbos MLJ, Heitman LH.Methods Cell Biol. 2019;149:179-194.
- Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor. Doornbos MLJ, Van der Linden I, Vereyken L, Tresadern G, IJzerman AP, Lavreysen H, Heitman LH.Biochem Pharmacol. 2018 Jun;152:201-210.
- Discovery and Kinetic Profiling of 7-Aryl-1,2,4-triazolo[4,3-a]pyridines: Positive Allosteric Modulators of the Metabotropic Glutamate Receptor 2. Doornbos MLJ, Cid JM, Haubrich J, Nunes A, van de Sande JW, Vermond SC, Mulder-Krieger T, Trabanco AA, Ahnaou A, Drinkenburg WH, Lavreysen H, Heitman LH, IJzerman AP, Tresadern G.J Med Chem. 2017 Aug 10;60(15):6704-6720.
Nuclear Hormone-Mediated Signaling
- Effects of exposure to six chemical ultraviolet filters commonly used in personal care products on motility of MCF-7 and MDA-MB-231 human breast cancer cells in vitro. Alamer M, Darbre PD.J Appl Toxicol. 2018 Feb;38(2):148-159.
- Anti-androgenic mechanisms of Bisphenol A involve androgen receptor signaling pathway.Wang H, Ding Z, Shi QM, Ge X, Wang HX, Li MX, Chen G, Wang Q, Ju Q, Zhang JP, Zhang MR, Xu LC.Toxicology. 2017 Jul 15;387:10-16.
- Toxicological effect of single contaminants and contaminant mixtures associated with plant ingredients in novel salmon feeds. Søfteland L, Kirwan JA, Hori TS, Størseth TR, Sommer U, Berntssen MH, Viant MR, Rise ML, Waagbø R, Torstensen BE, Booman M, Olsvik PA.Food Chem Toxicol. 2014 Nov;73:157-74.
- Real-Time Growth Kinetics Measuring Hormone Mimicry for ToxCast Chemicals in T-47D Human Ductal Carcinoma Cell. Daniel M. Rotroff, David J. Dix, Keith A. Houck, Robert J. Kavlock, Thomas B. Knudsen,Matthew T. Martin, David M. Reif, Ann M. Richard, Nisha S. Sipes, Yama A. Abassi, Can Jin, Melinda Stampfl, and Richard S. Judson. Chem. Res. Toxicol. 2013;26(7):1097–1107
- The Aryl Hydrocarbon Receptor Mediates Leflunomide- Induced Growth Inhibition of Melanoma Cells. Edmond F. O’Donnell, Prasad Rao Kopparapu, Daniel C. Koch, Hyo Sang Jang, Jessica Lynne Phillips, Robert L. Tanguay, Nancy I. Kerkvliet, Siva Kumar Kollur. PLoS One. 2012;7(7):e40926.
RTK-Mediated Cell Signaling
- Benzothiophene containing Rho kinase inhibitors: Efficacy in an animal model of glaucoma. Davis RL, Kahraman M, Prins TJ, Beaver Y, Cook TG, Cramp J, Cayanan CS, Gardiner EM,McLaughlin MA, Clark AF, Hellberg MR, Shiau AK, Noble SA, Borchardt AJ. Bioorganic & Medicinal Chemistry Letters. 2010 Jun 1;20(11):3361-6.
- Modeling ERBB receptor-regulated G1/S transition to find novel targets for de novo trastuzumab resistance. Sahin O, Fröhlich H, Löbke C, Korf U, Burmester S, Majety M, Mattern J, Schupp I,Chaouiya C, Thieffry D, Poustka A, Wiemann S, Beissbarth T, Arlt D. BMC Syst Biol. 2009 Jan 1;3:1.
- Label-free and real-time cell-based kinase assay for screening selective and potent receptor tyrosine kinase inhibitors using microelectronic sensor array. Atienza JM, Yu N, Wang X, Xu X, Abassi Y. J Biomol Screen. 2006 Sep;11(6):634-43.