Halotag ph sensor ligand

The following reagents were used: DAMGO (D-Ala(2)-N-Me-Phe(4)-Glyol(5)-enkephalin), forskolin, IBMX (3-isobutyl-1-methylxanthine), Ro 20-1724 (Sigma-Aldrich, Saint Louis, MO, USA), HaloTag^® pH Sensor Ligand (Promega, Madison, WI, USA), Hoechst 33342 (Dojinkagaku, Kumamoto, Japan), morphine hydrochloride (Takeda Pharmaceutical Co., Ltd., Tokyo, Japan), and FEN and REM (Janssen Pharmaceutical K. K., Tokyo, Japan). Forskolin, IBMX, Ro 20-1724, and all inhibitors were diluted with dimethyl sulfoxide, whereas the other reagents were diluted with H₂O.

To analyze KOR internalization, we used the membrane-impermeable Halotag^® pH sensor ligand, as previously described by Manabe et al. for MOR³⁷. Briefly, HEK293 cells stably expressing human KOR-fused Halotag^® were seeded at 9.0 $$\times$$ 10⁴ cells/well in an eight-chambered coverglass (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and incubated in 5% CO₂ at 37 °C for 21–24 h. The cells were washed with Hanks’ balanced salt solution and treated with 0.5 µM Halotag^® pH Sensor Ligand (Promega) at 37 °C for 15 min. The cells were washed again with Hanks’ balanced salt solution and treated with 4 µg/mL Hoechst 33342 (Dojindo Laboratories, Kumamoto, Japan) at 37 °C for 10 min. Red spots were observed using a Leica TCS SP8 lightning confocal microscope with a 40 $$\times$$ oil immersion lens (Leica, Wetzlar, Germany). After the baseline was measured for 10 min, images were obtained in each well every 10 min for 120 min after drug injection. The acquired images were analyzed for changes in the fluorescence intensity of total red spots/cells (the number of cells was defined by the number of nuclei as determined by Hoechst staining) with MetaMorph^® 7.7 (Molecular Devices) and were normalized to those before injection (% of before injection at 0 min).