Bovine albumin solution

Ob/Ob mice and B6 controls were injected subcutaneously with 40×10⁶ B16-FLt3-L-producing melanoma cells to in vivo expand the number of DCs in secondary lymphatic organs (Clement et al., 2016a (link)). Twelve to 14 days after injection spleens and lymph nodes (cervical, axillary, inguinal and mesenteric) were harvested and DCs were purified by density-gradient centrifugation using 30% Bovine Albumin solution (Sigma-Aldrich). Three mls of sterile BSA (30%) was mixed 1:1 (v/v) with RPMI1640 complete media on top of which an extra layer of 3 mL sterile BSA (30%) was added to complete the gradient premixing solution in a centrifuge polycarbonate tube that fits inside the Beckman SW Ti41 rotor. One-two mL of cell suspension was added on top of the premixed gradient solution and the mixture was centrifuged at 8500 rpm, 1 hour at 4°C using the Optima XPN-100k UltraCentrifuge (Bekman Coulter). The white ring of cells isolated after centrifugation routinely contains 70 to 80% CD11c+ DCs (Clement et al., 2016a (link)), with no significant differences observed in the numbers of cells isolated from control or Ob/Ob mice.