Cytoperm buffer

HEK293T cells were washed and surface stained at 4 °C with primary antibodies for SERINC5 and cell viability dye Zombie UV (Biolegend). For intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with CytoPerm buffer (Biolegend). Primary antibody anti-HA.11 epitope tag-PE (16B12, Biolegend) was used to detect SERINC5 and IFITMs. Data were acquired on a BD LSR Fortessa X-20 or Calibur cytometer and analyzed using FlowJo.

T cells were washed and incubated with antibodies (described above) at 4 °C for surface staining and cell viability using dye Zombie UV (Biolegend, San Diego, CA, USA). Primary antibodies were detected with anti-human IgG secondary antibody, fixed and analysed. To stain for intracellular antigens, cells were fixed with 4% paraformaldehyde and permeabilised with CytoPerm buffer (Biolegend, San Diego, CA, USA). Cells were analysed on BD LSR Fortessa X-20 cytometer (BD Biosciences Systems & Reagents Inc., San Jose, CA, USA). Compensation was performed using CompBeads (BD Biosciences Systems & Reagents Inc., San Jose, CA, USA) and calculated by FACSDiva software (BD Biosciences Systems & Reagents Inc., San Jose, CA, USA). Data were analysed using FlowJo software (BD Biosciences Systems & Reagents Inc., San Jose, CA, USA).