Fig. 2. (A) FACS analysis of the mean fluorescent intensity (MFI) of WT, hCB₂R, mCB₂R and hCB₁R overexpressing CHO cells at different concentrations of 8-SiR. (B) FACS analysis of the MFI of WT and hCB₂R-CHO cells pre-treated with JWH133 (10 μM) and stained with different concentrations of 8-SiR; see ESI† for details. (C and D) TR-FRET characterization of 8-SiR binding association (C) and saturation analysis (D) using HEK-hCB₂R cell membranes. (E and F) Time-lapse confocal microscopy frames for hCB₂R (E) and hCB₁R (F). CHO cells co-stained with 8-SiR (red) and Hoechst 33342 (cyan, nucleus counter stain) at 1, 4, 6, 8 and 10 min; plasma and internal membranes are highlighted with white and yellow dashes, respectively. (G) Association curve of 0.4 μM 8-SiR on plasma membrane and internal membranes of hCB₂R-CHO cells. See also ESI Videos S1 and S2.†.