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LUXendin645 (CELT-112) and GLP1R antibody labeling is not detectable in Glp1r(GE)−/− islets (scale bar = 40 µm) (n = 27 islets, five animals per genotype, three separate islet preparations). LUXendin645 (CELT-112) was applied at 100 nM.
Ligand-receptor binding and conformational rearrangement with GLP-1R agonists. (A) Schematic showing amino acid sequences of peptide ligands used in this study in single letter code. (B) Competitive binding of each ligand in competition with 10 nM Luxendin645 (CELT-112), n = 5, with calculated Ki shown and compared by one-way randomised block ANOVA with Tukey’s test. (C) As for (B) but with 5 nM exendin-4-Cy5 (Ex4-Cy5) as the competing ligand, n = 5. (D) Principle of NR12A assay (cartoon generated using BioRender), showing how ligand-induced Lumi4-Tb-labelled SNAP-GLP-1R ECD movement leads to an increase in energy transfer to the plasma membrane probe NR12A, detected as an increase in TR-FRET. (E) The TR-FRET spectrum of Lumi4-Tb-labelled HEK293-SNAP-GLP-1R cells with and without co-labelling with 100 nM NR12A, n = 2. (F) ECD movement response with NR12A + Lumi4-Tb-labelled HEK293-SNAP-GLP-1R cells stimulated with exendin-4, exendin-F1 or P5, n = 4. Kinetic and concentration responses are displayed. *p < 0.05 by indicated statistical test. Data are represented as mean ± SEM, with individual replicates shown where possible.
a. Exendin4(9–39), S39C-Exendin4(9–39), and LUXendin645 (CELT-112) display similar antagonistic properties (applied at 1 µM) in HEK293-SNAP_GLP1R following 30 min GLP-1-stimulation (n = 4 independent assays). b. LUXendin645 (CELT-112) weakly activates GLP1R in the presence of the positive allosteric modulator (PAM) BETP (25 µM) (30 min stimulation in HEK293-SNAP_GLP1R) (Ex4, +ve control) (n = 4 independent assays). c. LUXendin645 (CELT-112) labels AD293-SNAP_GLP1R cells with maximal labeling at 250–500 nM (n = 4 independent assays). d. LUXendin645 (CELT-112) signal cannot be detected in YFP-AD293 cells (scale bar = 212.5 µm) (n = 3 independent assays). e. Representative confocal z-stack showing LUXendin645 (CELT-112) staining in a live islet (n = 27 islets, six animals, three separate islet preparations) (scale bar = 37.5 µm). f. As for e, but two-photon z-stack (scale bar = 37.5 µm) (representative image from n = 27 islets, seven animals, three separate islet preparations). g, h. 250 nM LUXendin645 (CELT-112) internalizes GLP1R in MIN6 β-cells when agonist activity is conferred using 25 µM BETP (Ex4 and Ex9 were applied at 100 and 250 nM, respectively) (scale bar = 21 µm) (representative images from n = 12 coverslips, three independent repeats) (one-way ANOVA with Bonferroni’s test; F = 217.6, DF = 3). i, j. LUXendin645 (CELT-112) signal co-localizes with a GLP1R monoclonal antibody in islets (n = 13 islets, three separate islet preparations) and MIN6 β-cells (representative images from n = 24 coverslips, three independent repeats) (scale bar = 26 µm). k. LUXendin645 (CELT-112) improves membrane visualization compared to antibody (scale bar = 12.5 µm). Representative images are shown, with location of intensity-over-distance measures indicated in blue (n = 18 islets, five animals, three separate islet preparations). l, m. LUXendin645 (CELT-112) co-localizes with Surface 488, pre-applied to Glp1r null SNAP_hGLP1R-INS1GLP1−/− cells l. Pre-treatment with Exendin4(1–39) to internalize the GLP1R reduces LUXendin645 (CELT-112)–labeling m. (scale bar = 10 µm) (representative images from n = 3 independent repeats). LUXendin645 (CELT-112) was applied to cells at 250 nM and tissue at 50–100 nM. GLP-1 glucagon-like peptide-1; Ex9 Exendin4(9–39); S39C S39C-Exendin4(9–39); Ex4 Exendin4(1–39).
a–c. LUXendin645 (CELT-112) labeling is widespread throughout the intact islet, co-localizing predominantly with β-cells a and δ-cells b, but less so with α-cells c stained for insulin (INS), somatostatin (SST), and glucagon (GCG), respectively (n = 18 islets, seven animals, three separate islet preparations) (scale bar = 26 µm). d. Following dissociation of islets into cell clusters, LUXendin645 (CELT-112) labeling can be more accurately quantified (arrows highlight cells selected for zoom-in) (scale bar = 26 µm). e. Zoom-in of d showing a LUXendin645 (CELT-112)− (left) and LUXendin645 (CELT-112)+ (right) α-cell (arrows highlight non-labeled cell membrane, which is not bounded by a β-cell) (scale bar = 26 µm). f. Box-and-whiskers plot showing proportion of β-cells (INS) and α-cells (GCG) co-localized with LUXendin645 (CELT-112) (n = 18 cell clusters, ten animals, three separate islet preparations) (box and whiskers plot shows range and median; mean is shown by a plus symbol). g. Ins1CreThor;R26mT/mG dual fluorophore reporter islets express tdTomato until Cre-mediated replacement with mGFP, allowing identification of β-cells (~80% of the islet population) and non-β-cells for live imaging (scale bar = 26 µm). LUXendin645 (CELT-112) highlights GLP1R expression in nearly all β-cells but relatively few non-β-cells (n = 31 islets, six animals, three separate islet preparations). h. A zoom-in of the islet in g showing GLP1R expression in some non-β-cells (left) together with quantification (right) (arrows show LUXendin645 (CELT-112)-labeled non-β cells) (scale bar = 12.5 µm) (scatter dot plot shows mean ± s.e.m.). White boxes show the location of zoom-ins. In all cases, LUXendin645 (CELT-112) was applied at 100 nM.
LUXendin645 (CELT-112) allows super-resolution snapshots of MIN6 β-cells using widefield microscopy combined with super-resolution radial fluctuations (SRRF) (representative image from n = 8 images, three independent repeatss) (scale bar = 10 µm for full-field images, 2.5 µm for zoomed-in images). White boxes show the location of zoom-ins. The following compound concentrations were used: 100 nM LUXendin645 (CELT-112) (SRRF).
a. LUXendin645 (CELT-112) labeling is detected in the the median eminence (ME), arcuate nucleus (ARC), area postrema (AP)/nucleus tractus solitaris (NTS), and choroid plexus (CP), in close association with GLP1-producing neurons, identified using GLU-YFP reporter animals (3V, third ventricle) (representative images from n = 4 animals) (scale bar = 106 µm). b. Z-projection of an image stack (~30 µm) showing direct contacts between LUXendin645 (CELT-112)-labelled and GLP1-producing (GLU-YFP) neurons in the ARC (representative image from n = 4 animals) (scale bar = 20 µm). c, d. LUXendin645 (CELT-112) labeling co-localizes with GLP1R+ neurons in the AP/NTS c and ARC d, identified using GLP1RCre;LSL-GCaMP3 reporter animals (representative image from n = 4 animals) (scale bar = 61 µm). e. Super-resolution imaging using Airyscan shows that LUXendin645 (CELT-112) labeling is restricted to the membrane of the cell body and dendrites of GLP1R+ neurons (arrows show cell body and dendrite from left to right, respectively) (representative images from n = 4 animals) (scale bar = 9 µm). f. GLP1R form nanodomains in ARC and AP neurons, as well as ependymal cells of the CP (confocal image is shown on the left for comparison) (representative images from n = 8 animals) (scale bar = 9 µm). g. Mapping of LUXendin645 (CELT-112) distribution in cleared brains shows labelling of the ARC, AP/NTS, CP, lateral ventricles (LV), fourth ventricle (4V), subfornical organ (SFO), and organum vasculosum of the lamina terminalis (OVLT) (representative images from n = 4 animals) (scale bar = 1 mm). Note that, due to suspension of the brain, the coronal section is slightly offset in the dorsal–ventral plane; hence, the SFO appears above the ARC. In all cases, LUXendin645 (CELT-112) was injected subcutaneously at 100 pmol/g.
a. LUXendin645 (CELT-112) labels β-like cells in intact spheroids, which were differentiated and cultured for 21 days. No signal is detected in undifferentiated human ES cells (day 0) or unlabelled β-like cells (-LUXendin645 (CELT-112)) (representative images from n = 6 spheroids) (scale bar = 100 µm). b. GLP1R gene expression in day 0 undifferentiated cells, day 21 differentiated β-like cells, and human islets (n = 3 donors). c. LUXendin645 (CELT-112) labelling is localized to strongly insulin (INS)-positive but not strongly glucagon (GCG)-positive areas (representative images from n = 5–6 spheroids) (scale bar = 50 µm). d. LUXendin645 (CELT-112) overlaps more with INS vs. GCG, as calculated using Manders’ M1 co-efficient (n = 5–6 spheroids) (unpaired Student’s t-test). e. Day 21 spheroid sections (5 µm) showing expression of INS and NKX6-1, confirming a differentiated phenotype (representative images from n = 4 spheroids) (scale bar = 100 µm). f. FACS plots of day 21 β-like cells with and without LUXendin645 (CELT-112) incubation. LUXendin645+ (LUX+) and LUXendin645− (LUX−) cells were sorted for qPCR. g. GLP1R, NKX6-1, INS, and GCG gene expression in sorted cells (n = 4 spheroids) (connecting bars indicate LUX+ and LUX− populations in the same samples) (paired Student’s t-test). LUXendin645 (CELT-112) was applied at 100 nM.

Our GLP1R fluorescent antagonist shows high affinity for GLP1 receptor (pEC50 = 7.5 for GLP1R) modulating the receptor by orthosteric antagonism. The efficacy and potency of LUXendin645 (CELT-112) as a GLP1R ligand was confirmed by inhibition of GLP-1-stimulated cAMP levels in SNAP-GLP1R:HEK293 cells. LUXendin645 (CELT-112) has been used in a variety of imaging applications, including widefield/confocal/2-photon microscopy in live and fixed mammalian cells and tissue. Using TR-FRET, LUXendin645 was used in GLP1R competitive binding experiments and in GLP1R trafficking and recycling studies.

 

More information and applications

Article 1: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics. Nature Communications (2020).

Article 2: Evaluation of efficacy- versus affinity-driven agonism with biased GLP-1R ligands P5 and exendin-F1. Biochem Pharmacol. (2021).

Article 3: Glucagon-Like Peptide-1 Receptor Regulates Thromboxane-Induced Human Platelet Activation. JACC Journals (2022).

Article 4: Revealing the tissue-level complexity of endogenous glucagon-like peptide-1 receptor expression and signaling. Nature Communications (2023).

Article 5: A neuronal circuit driven by GLP-1 in the olfactory bulb regulates insulin secretion. Nature Communications (2024).

Article 6: Localized GLP1 receptor pre-internalization directs pancreatic alpha cell to beta cell communication. Cell Metabolism (2025).

LUXendin645 GLP1 receptor (GLP1R) fluorescent antagonist (645/664) (CELT-112)
For research use only

3 nmol vial that allows to prepare 30 µL of 100 µM stock solution to test GLP1R. One vial is sufficient for injecting one mouse.

Please note that the required concentration may vary depending on the specific assay design.

424,00€

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