Material & Methods
Reagents: SNAP-Lumi4-Tb, antimouse Fc-d2, SNAP-CXCR4, and SNAP-EGFR plasmids and the labeling medium were from Cisbio Bioassays. Lipofectamin 2000 was purchased from Invitrogen, and the cell-dissociation buffer was from Sigma Aldrich. Anti-CXCR4 antibodies (12G5 and 44717) and anti-CXCR7 antibody (11G8) were from R&D Systems.
Covalent labeling of SNAP-receptor cells: A solution of Tag-lite SNAP-Lumi4-Tb substrate at 100 nM was prepared in Tag-lite labeling medium. After aspiration of the cell culture medium, 3 mL of this solution was added to the cells expressing the SNAP-receptor in the T175 flask, followed by a one hour incubation at 37ºC. The cells were then washed four times to remove the excess of SNAP-Lumi4-Tb and detached using the cell dissociation buffer. The cells were frozen under 1 million cells/vial in culture medium and 10% DMSO.
Ligand binding assay: The SNAP-Lumi4-Tb labeled cells were thawed and washed with PBS, then resuspended in labeling buffer. Binding assays were performed in a total volume of 20 µL in 384-small volume white plates. 10,000 cells/well were incubated for one hour at room temperature with different concentrations (0–200 nM) of labeled ligand (SDF1-red, mAb anti-CXCR4-red) or unlabeled ligand (mAbs anti-CXCR4). 100 nM of antimouse Fc-d2 was added for the detection of unlabeled mAbs.
Competition binding assay: Competition binding assays were carried out in a final volume of 20 µL in 384-small volume white plates. 10,000 labeled cells were incubated with red-labeled SDF1 or red-labeled EGF (final concentration of 12.5 nM or 10 nM respectively) in the presence of different concentrations of antibodies (0–100 nM). Following incubation, the plates were measured on a TR-FRET reader.
cAMP functional assay with cAMP dynamic 2 kit: The functional tests were performed in 20 µL final volume in a 384-small volume plate. 10,000 cells were incubated with Forskolin (2 µM final concentration) and different concentrations of SDF1 or 12G5 antibody (0–1 µM final concentration). The plates were incubated 30 minutes at room temperature before being read on a TR-FRET reader.
Internalization Assay: Internalization assays were carried out in a final volume of 20 µL in 384-small volume white plates. 10,000 labeled cells were incubated at room temperature with a high concentration of internalization reagent in the presence or not of different concentrations of SDF1. The plate was measured after a two-hour incubation.