CHO cells expressing SNAP-tagged membrane receptors were washed twice with phosphate-buffered saline (PBS). Covalent labeling of the SNAP-tag was conducted by incubating the cells for 20 min at 37°C, 5% (v/v) CO2 with 2 μM of the membrane-impermeable SNAP substrate SNAP-Surface Alexa Fluor 647 diluted in complete phenol red–free culture medium. At the end of the incubation, the cells were washed three times with complete phenol red–free medium, with 5-min incubation at 37°C, 5% (v/v) CO2 after each wash. Last, the cells were washed twice with PBS for 5 min at room temperature (RT) and fixed with 4% paraformaldehyde (PFA) for 15 min at RT.

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