A 11094

Cells were seeded, transfected and resuspended as described above. The antibody uptake assay was based on the use of quenching to distinguish internal from external material^{68 (link)}. Approximately 10⁶ cells were resuspended in complete medium containing an anti-HA AF488 conjugate (1:1000) and incubated at 37 °C for 40 min. The cells were washed twice with ice cold FACS buffer and incubated with an anti-AF488 antibody (1:67, A-11094, Thermo Fischer Scientific; to quench non-internalised anti-HA AF488 conjugate), an anti-mouse AF647 antibody (1:300, Thermo Fischer Scientific, A31571; to relabel the non-internalised anti-HA conjugate) and an eFluor 780 fixable viability dye. Cells were washed three times in ice cold FACS buffer, fixed in 4% paraformaldehyde (PFA) for 20 min and washed a further two times in FACS buffer. Cells were strained and analysed as described above. Internalised anti-HA AF488 conjugate was inaccessible to quenching and therefore any associated AF488 signal was equated to levels of internalised S. Conversely, only anti-HA AF488 conjugate at the cell surface was accessible by the anti-mouse AF647 secondary antibody and therefore any associated AF647 signal was equated to levels of non-internalised S.