5 × 104 cells were fixed in 4% formaldehyde (Sigma Aldrich, St. Louis, MO, USA) and immunostained for 45 min on ice in PBS containing 0.5% bovine serum albumin (PBS-0.5% BSA). The antibodies used, were PE-conjugated anti-human CXCR4 (clone 1D9, BD, Franklin Lakes, NJ, USA), biotinylated anti-mouse CXCR4 (clone 2B11, BD, Franklin Lakes, NJ, USA) and APC-conjugated anti-human/mouse CCR7 (clone 3D12, eBioscience, Thermo Fisher Scientific, Waltham, MA, USA). Samples containing biotinylated antibodies were treated with PE-conjugated streptavidin (Rockland Immunochemicals, Limerick, PA, USA) in PBS/0.5% BSA for 30 min on ice. Flow cytometry was carried out using FACSCanto equipment (BD, Franklin Lakes, NJ, USA) with standard settings. Data analysis was performed using FlowJo software (BD, Franklin Lakes, NJ, USA). Positive events were defined above the level of background staining observed using matched isotype control antibodies.
For the FACS-FRET experiments, the bandpass filter settings for detection on FACSCanto equipment were changed to the following excitation/emission windows: PE—488 nm/>556LP + 585 ± 42; FRET—488 nm/>655 nm, and APC—633 nm/660 ± 20. Positive FRET signal was defined as the level of fluorescence above background staining observed using matched isotype control antibodies.
For the FACS-FRET experiments, the bandpass filter settings for detection on FACSCanto equipment were changed to the following excitation/emission windows: PE—488 nm/>556LP + 585 ± 42; FRET—488 nm/>655 nm, and APC—633 nm/660 ± 20. Positive FRET signal was defined as the level of fluorescence above background staining observed using matched isotype control antibodies.