Bone Marrow Cells Stimulation Protocol

Bone marrow cells were cultured as described previously [47 (link),48 (link)]. In brief, bone marrow cells from Cnr1^−/−, Cnr2^−/− and Cnr1^−/−/Cnr2^−/− mice were seeded at 5 × 10⁵ cells/mL in DMEM supplemented with 10% FCS, 1% MEM/NEAA, 1% Penicillin/Streptavidin 0.1% β-Mercaptoethanol (all from Thermo Fisher Scientific, Inchinnan, UK) and 10% conditioned medium of B16 cells. Medium was exchanged on day 3 and cells stimulated on day 5 with either 100 ng/mL Escherichia coli Lipopolysaccharide (LPS) Serotype 0127:B8 (Sigma-Aldrich, Saint Louis, MO, USA) or 1 nmol/mL CpG1668 (TIB MOLBIOL, Berlin, Germany). After 16 h of stimulation, cells were harvested and analyzed by FACS or transferred in the migration assay.