Covalent Labeling of Reovirus Capsid

Reovirus particles were randomly labeled at the lysine side chains on the viral capsid^{57 (link)}. Therefore, reoviruses were first dialyzed against PBS to remove free amines in Slide-A-Lyzer MINI dialysis Devices, 3500 MWCO (Thermo Fischer Scientific, USA) at 4 °C overnight. Then 100 μl of the virus solution were mixed with the desired NHS-ligand and incubated at RT for 1 h. For experiments with covalently immobilized reovirus on single tension probes, propargyl-NHS at 33.2 μM final concentration and Alexa568-NHS (Thermo Fisher Scientific, USA) at 0.5 μM final concentration (for TIRF imaging) or Alexa647-NHS (Thermo Fisher Scientific, USA) at 32 μM final concentration (for fluorescence widefield and confocal imaging), and for experiments with non-covalently immobilized reovirus on neutravidin surfaces, 8.4 μM EZ-link sulfo-NHS-biotin and Alexa647-NHS at 33.7 μM final concentration (both Thermo Fisher Scientific, USA) were used. Particles were purified by gel filtration with Zeba spin columns (7000 MWCO, Thermo Fisher Scientific, USA).