GLP-1R internalization and recycling in CHO-SNAP-GLP-1R cells was measured by surface labeling with anti-GLP-1R antibody or the SNAP-tag probe Lumi4-Tb (Cisbio). For antibody labeling, after treatments at 37 °C, cells were placed on ice to arrest further endocytosis before fixation, and the surface GLP-1R was detected by ELISA with monoclonal anti-human GLP-1R antibody68 (link) (Mab 3F52, Developmental Studies Hybridoma Bank (DSHB), 1/100) plus horseradish peroxidase (HRP)-conjugated rabbit anti-mouse secondary (ab97046, Abcam, 1/5,000). 3,3′,5,5′- tetramethylbenzidine (TMB) substrate was added and the absorbance was read at 450 nm after 1 M HCl addition. For Lumi4-Tb, cells were labeled at 40 nM at 4 °C, followed by time-resolved (TR) fluorescence measurement in a Molecular Devices i3x (excitation 335 nm, emission 620 nm). Residual surface expression was determined from peptide- vs. control-treated wells. Recycling was measured by comparing the surface receptor immediately after internalization vs. a further period of recycling with agonist washed off and replaced with 10 μM exendin(9-39); and recycling calculated as percentage recovery of surface GLP-1R in the recycling plate vs. surface receptor loss in the internalization plate.
Lumi4 tb
Manufactured by PerkinElmer
Sourced in France, United States, United Kingdom
The Lumi4-Tb is a high-performance luminescent lanthanide chelate designed for use in time-resolved fluorescence (TRF) assays. It serves as a stable and sensitive label for biological molecules, enabling efficient energy transfer and long-lived fluorescence emission.
Automatically generated - may contain errors
Lab products found in correlation
Ab97046, by Abcam (1 mentions)
Mab 3f52, by Developmental Studies Hybridoma Bank (1 mentions)
Snap red, by PerkinElmer (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Enzyme free cell dissociation buffer, by Merck Group (1 mentions)
Black 384 well plate, by Greiner (1 mentions)
Hbss buffer, by Thermo Fisher Scientific (1 mentions)
Schrodinger software, by Schrödinger (1 mentions)
Tag lite buffer, by PerkinElmer (1 mentions)
Lumi4 tb, by Tecan (1 mentions)
8 protocols using lumi4 tb
1
Measuring GLP-1R Internalization and Recycling
2
Time-Resolved FRET Analysis of SNAP-β2AR
3
SNAP-tagged Protein Fluorescence Labeling
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SNAP-tagged proteins (ACE2, LepR, VEGFR2) were fluorescently labeled by incubating cells transfected with the corresponding expression vectors with an SNAP suicide substrate conjugated to the long-lived fluorophore Terbium cryptate (Tb; Lumi4-Tb, 100 nM; Cisbio Bioassays) in Tag-lite labeling medium (1 h, on ice) (Keppler et al., 2003 (link)). After several washes, cells were collected using enzyme-free cell dissociation buffer (Sigma-Aldrich), resuspended in Tag-lite buffer and distributed into a 384-well plate. Efficient fluorescent labeling of SNAP was verified by reading fluorescence signal at 620 nm. Experiments were then conducted according to the different modes described below. All reagents were diluted in Tag-lite buffer (final reaction volume of 14 μL) and incubations were performed at room temperature; except in the case of compounds Fenbendazole and Elaidic Acid, where incubation was performed at 37°C to avoid precipitation of compounds. TR-FRET signals were detected using a plate reader (Tecan F500; Tecan, Männedorf, Switzerland) with the following settings: excitation at 340 nm (Tb, energy donor), emission at 665 nm (d2, acceptor) and 620 nm (donor); delay of 150 μs; and integration time of 500 μs. Data is expressed as TR-FRET ratio (acceptor/donor). When indicated, TR-FRET ratio was normalized to % of basal or % of maximal binding (Bmax).
4
Characterization of Secretin Analogs
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Human secretin, human glucagon, and secretin analogs 1–5 and 15–20 of greater than 95% purity were purchased from GenScript, USA. The SNAP-tag vector (PLASCUST), Tag-lite® labeling media (SSNPTBX), and Lumi4-Tb (SSNPTBD) were purchased from Cisbio, USA. MEM media (61100–061), Versene (15040–066) and HBSS buffer (14025134) were purchased from Gibco®, Life Technologies. Primers were custom designed and purchased from Invitrogen. The HTRF-LANCE® cAMP assay kit (AD0262) was obtained from PerkinElmer. The 384-well black plates were purchased from (Greiner Bio-One, 788086). The MOE software was licensed through Cloud Scientifics, China. Schrodinger software was licensed from Schrödinger LLC.
5
GPCR Cell Surface Expression Quantification
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On day 0, HEK293 cells were seeded at 5 × 105 cells per 60 mm dish. On day 1, cells were transfected with 0.5 µg/dish of SNAP-tagged GPCRs. Following treatments described in the figure legends, for experiments shown in Figure 1B cells were treated with 5 μM cell-impermeable (SNAP-Surface 488) or cell–permeable (SNAP-Cell 505 Star) SNAP-tag substrate. For experiments shown in Figure 2D , cells were treated with 100 nM cell-impermeable substrate Lumi4-Tb (CisBio, Bedford, MA). The labeling reaction was carried out for 1 hr at 37°C in 8% CO2. Labeled cells were washed in Tag-Lite Buffer (CisBio) and resuspended to 7.5 × 105 cells/ml. Cell suspension (100 μl) were added in triplicate to a 96-well dish and fluorescence was measured using a Tecan plate reader with excitation and emission of 506 and 526 nm for SNAP-Surface 488 and SNAP-Cell 505 Star or an excitation and emission of 340 and 620 nm for Lumi4-Tb, respectively.
6
SNAP-ACE2 Receptor Binding Assay
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Assay was performed as recently described [[41] (link), [50] ]. Briefly, HEK293 cells were transiently transfected to express SNAP-tagged ACE2 construct, which was fluorescently labelled by incubating cells with a SNAP suicide substrate conjugated to the long‐lived fluorophore Terbium cryptate (Tb; Lumi4‐Tb, 100 nM; Cisbio Bioassays). Labelled cells were collected and re-distributed into a 384-well plate. Cells were pre-incubated (1 h) with test compounds at the indicated concentration followed by addition of RBD-d2 (2 h, room temperature). TR-FRET signals were detected using a plate reader (Tecan F500; Tecan, Männedorf, Switzerland) with the following settings: excitation at 340 nm (Tb, energy donor), emission at 665 nm (d2, acceptor) and 620 nm (donor). Data is expressed as TR-FRET ratio (acceptor/donor) and normalized to % of basal or % of control group.
7
Monitoring Receptor Internalization with DERET
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DERET (8 (link)) was used to monitor agonist-induced receptor internalization in HEK293T cells transiently transfected for 24 h with SNAP-tagged receptors (2 μg plasmid DNA per well of 6-well plate) or in monoclonal CHO-K1 cells stably expressing SNAP-GLP-1R or SNAP-GIPR. Labeling was performed using the time-resolved Förster resonance energy transfer SNAP-probe Lumi4-Tb (Cisbio) at 40 nM for 60 min at room temperature, either in suspension (for HEK293T) or with adherent cells (for CHO-K1). After washing three times, fluorescein (24 μM in HBSS) was added to cells in opaque bottom white plates, and baseline signal was read for 10 min using a Flexstation 3 plate reader (λex 340 nm, λem 520 and 620 nm, delay 400 μs, integration 1500 μs) at 37 °C. Agonists, prepared in 24 μM fluorescein, were added, and signal was sequentially monitored. Receptor endocytosis leads to reduced contact of Lumi4-Tb with extracellular fluorescein, and a reduction in signal at 520 nm with an increase at 620 nm. After first subtracting values from wells containing fluorescein only, internalization was expressed ratiometrically as signal obtained at 620 nm divided by that obtained at 520 nm.
8
Monitoring Receptor Internalization by DERET
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Diffusion-enhanced resonance energy transfer (DERET) (7) was used to monitor agonistinduced receptor internalisation in HEK293T cells transiently transfected for 24 hours with SNAP-tagged receptors (2 µg plasmid DNA per well of 6-well plate), or in monoclonal CHO-K1 cells stably expressing SNAP-GLP-1R or SNAP-GIPR. Labelling was performed using the timeresolved Förster resonance energy transfer (TR-FRET) SNAP-probe Lumi4-Tb (Cisbio) at 40 nM for 60 minutes at room temperature, either in suspension (for HEK293T) or with adherent cells (for CHO-K1). After washing three times, fluorescein (24 µM in HBSS) was added to cells in opaque bottom white plates, and baseline signal was read for 10 min using a Flexstation 3 plate reader (λex 340 nm, λem 520 and 620 nm, delay 400 µs, integration 1500 µs) at 37°C. Agonists, prepared in 24 µM fluorescein, were added, and signal was sequentially monitored. Receptor endocytosis leads to reduced contact of Lumi4-Tb with extracellular fluorescein, and a reduction in signal at 520 nm with an increase at 620 nm. After first subtracting values from wells containing fluorescein only, internalisation was expressed ratiometrically as signal obtained at 620 nm divided by that obtained at 520 nm.
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