Tag lite buffer

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.

CHO cells were transfected according to the manufacturer's recommendation (jetPEI DNA transfection, Polyplus-transfection, Illkirch, France). Briefly, cells were seeded on day 1 in 6-well plates at a concentration of 300,000 cells/well. On day 2, 6 μl/well of JetPEI diluted in 100 μl NaCl (150 mM) were added to a mix of DNA coding for SNAP-OXTR (180 ng/well) and uncoding DNA (2820 ng /well) diluted in 100 μl NaCl (150 mM). The mixture was incubated for at least 30 min at room temperature and was then added onto the cells. On day 3, cells in the 6-well plates were then harvested after addition of trypsin, counted and seeded at a concentration of 30,000 cells/well in a white 96-well plates. Experiments were carried out on day 4. Cells were labeled with SNAP-Lumi4-Tb (100 nM) (Cisbio Bioassays, Codolet, France) at 37°C for 1 hour, rinsed four times with Tag-lite buffer (Cisbio Bioassays) and incubated in the presence of the various compounds. The time-resolved FRET (TR-FRET) signal was measured on a Pherastar (BMG Labtech). Cells were illuminated at 337 nm, and luminescent signals were measured at 620 nm and 665 nm every minute. The ratio (665/620) was then plotted as a function of time. Experiments were performed three times, independently.

The buffer (Tag-lite Buffer), the labeling dye (Lumi4-Terbium), the d2-coupled SDF1/CXCL12, SNAP-CXCR4 expression vector were all obtained from Cisbio Bioassays, Codolet, France. HEK293 FT cells were transfected with the SNAP-tagged hCXCR4. After 48 h of expression, cells were labeled with Lumi4-Terbium and seeded at 20,000 cells per well in 384-well plates. Cells were exposed to Nb at concentrations from 1 pM to 10 µM for 1 h before adding 12 nM of d2-coupled SDF1. Plates were read on a Mithras² LB 943 Monochromator Multimode Reader (Berthold Technologies, Thoiry, France).