ACKR3 Expression in Sf9 Cells

ACKR3 was expressed in Sf9 cells as previously described (27 (link)). Briefly, cells were cultured in ESF921 media (Expression Systems) at 27°C with shaking at 140 rpm. The Bac-to-Bac Baculovirus Expression System (Invitrogen) was used to produce baculovirus containing the different ACKR3 and CXCL12 variants as previously described (27 (link)). Briefly, recombinant bacmids were incubated with 3 μL of Xtreme Gene Transfection Reagent (Roche) and 100 μL of transfection medium (Expression Systems) for 30 minutes. The mixture was added to 2.5 mL of Sf9 cells at a density of 1.3 × 10⁶ cells mL⁻¹ and the cells were incubated for 96 h with 300 rpm shaking at 27°C. Cells were pelleted using centrifugation and 400 μL of the resulting supernatant (P0 stock) was used to transfect 40 mL of Sf9 cells at a density of 2.5 × 10⁶ cells mL⁻¹. After 48 h the cells were centrifuged and the resulting supernatant was stored at 4°C until further use (P1 stock). Virus titers were determined using flow cytometry staining with a phycoerythrin (PE)conjugated GP64 antibody. To initiate protein expression P1 virus corresponding to ACKR3 alone or ACKR3 and CXCL12 (co-expression) was added to Sf9 cells at a density of ~2.5 × 10⁶ cells mL⁻¹ at a multiplicity of infection of 5. Cells were grown for 48h at 27°C with shaking at 140 rpm prior to experiments.