BG-JF549

Guidelines (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).

1. Stock solution preparation
1.1 Solvent: anhydrous DMSO.
1.2 Stock solution concentration: 1 mM.

2. Working solution preparation
2.1 Working concentration: 500 nM in culture medium for live pancreatic islets; 30 nM for labeling HEK293-FLP cells; 1 μM in EX for labeling primary mouse striatal neurons and adherent HEK 293T cells expressing SNAP-tagged mGluR.

3. Staining procedure
3.1 Live pancreatic islets^[1]: Use live pancreatic islets isolated from GLP1R^SNAP/SNAP mice. Incubate the islets with 500 nM BG-JF549 in culture medium at 37°C, 5% CO₂ for 30 minutes. For immunostaining, fix the labeled islets in 4% formaldehyde for 12-15 minutes before incubation with monoclonal anti-GLP1R antibody.

3.2 HEK293-FLP cells expressing SNAP-MOR^[2]: Incubate with 30 nM BG-JF549 (SNAP-JF549) at 37°C for 30 minutes. Wash twice with PBS.

3.3 Primary mouse striatal neurons labeled with SNAP-MOR^[2]: Incubate with 1 μM BG-JF549 (SNAP-JF549) at 37°C for 15 minutes. If using SNAP-Surface Block, pre-label with 1 μM SNAP-Surface Block in at 37°C for 15 minutes before labeling, wash once with PBS after pre-labeling, then perform BG-JF549 labeling, and wash once with PBS after labeling.

3.4 Adherent HEK 293T cells expressing SNAP-tagged mGluR^[3]: Incubate the cells with 1 μM BG-JF549 in EX at 37°C for 45 minutes. Wash the cells after labeling.

4. Controls
4.1 Islet negative control: Process GLP1R^WT/WT islets in parallel with GLP1R^SNAP/SNAP islets.
4.2 mGluR cell negative control: Before BG-JF549 labeling, treat the cells with saturating antagonist in culture medium at 37°C for 1 hour; use 10 μM LY34 for mGluR2 or mGluR3, and 20 μM CPPG for mGluR8.

5. Detection and analysis
5.1 Instrument types: Airyscan confocal microscope, spinning disk confocal microscope; live-cell fluorescence microscope with widefield imaging.
5.2 Wavelength settings: Excite with a 561-nm laser; detect at 569-649 nm. SNAP-JF549 fluorescence is red.
5.3 Result analysis: In GLP1R^SNAP/SNAP islets, bright membrane-localized fluorescence is expected, with narrow fluorescence peaks around the cell membrane; no specific signal is expected in GLP1R^WT/WT islets, and in fixed islets the BG-JF549 signal should co-localize with GLP1R monoclonal antibody staining.
5.4 Result analysis: SNAP-JF549-labeled SNAP-MOR is detected on the cell surface and in intracellular vesicles, the signal translocates intracellularly after agonist exposure, and SNAP-MOR co-localized with LAMP1 appears yellow.
5.5 Result analysis: Measure the mean fluorescence intensity. A glutamate-induced decrease in total fluorescence indicates receptor degradation.