Rea649

Cells were transferred to FACS tubes (adherent cells were harvested using FACS buffer (PBS pH 7.4 with 0.05% BSA and 2 mM EDTA) prior to staining) with 1 mL of FACS buffer and centrifuged at 150 g for 5 min. Supernatant was decanted, and cells were resuspended in 50 μL of FACS buffer. 10 μL of human IgG (Thermo Fisher 027102) was added, cells were flicked to mix, and were incubated at 4°C for 5 min. Conjugated primary antibody was then added at the manufacturer's recommended dilution, cells were flicked to mix and incubated at 4°C for 30 min. Cells were then washed three times with 1 mL of FACS buffer, centrifuging at 150 g for 5 min and decanting the supernatant after each wash. Cells were resuspended in two drops of FACS buffer prior to flow cytometry. For Miltenyi Biotec antibodies, cells were stained at 4°C for 15 min without blocking and were washed once prior to flow cytometry, as per manufacturer protocol. Antibodies used in this study were as follows: Anti-FLAG-APC (Abcam ab72569), anti-CD2-APC (R&D Systems FAB18561A), anti-CXCR4 (Miltenyi REA649, 130-117-354), anti-CD25-PE (Miltenyi REA945, 130-115-628), anti-SLAM-PE (Miltenyi REA151, 130-123-970), and anti-mouse IgG1-APC (R&D Systems IC002A) or anti-human IgG1-PE (Miltenyi REA293, 130-113-438) were used as isotype controls where appropriate.