Dmem culture medium

Human embryonic kidney 293 (HEK-293) from the American Type Culture Collection (ATCC; CRL-1573^™) were grown in 75 cm² cell culture flasks (430,725; Corning, Barcelona, Spain) in DMEM culture medium (ATCC, 30–2002), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, Gibco, Life Technologies S.A., Madrid, Spain). When approaching 70–80% confluence, cells were harvested using trypsin–EDTA solution (25,300–054, Gibco, Barcelona, Spain) and transferred to 12-well plates containing poly-d-lysine coated glass coverslips. When they reached 70–80% confluence, cells were transfected with pcDNA3.0 plasmid containing a cDNA insert encoding the human cannabinoid receptor 1 (pCDNA-CB₁; 1 µg DNA/well) using Lipofectamine 3000 (L3000001; Invitrogen S.A., Spain). Cells were processed for single or double immunofluorescence 48 h after transfection.

The mouse macrophage cell line RAW 264.7 (ATCC, USA) was grown in DMEM culture medium (ATCC, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) in a 5% CO₂ humidified incubator at 37°C.