Ham s f 12 k media

CHO-K1 cells stably expressing wild-type recombinant hCB1Rs (CNR1) were purchased from DiscoverRx Corporation (Fremont, CA, United States) and designated as CHO-hCB1. Transfected cells were cultured in HAM’s F-12 K media (ATCC, Manassas, VA, United States) that also contained 10% fetal calf serum (Gemini Bio Products, Sacramento, CA, United States), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States), and 250 μg/mL of G418 (Sigma-Aldrich, St. Louis, MO, United States) in a humidified chamber at 37°C with 5% CO₂. Cells were harvested when flasks reached approximately 70% confluence, and only cells from passages 4–15 were used in all experiments.

Chinese hamster ovary (CHO-K1) cells were stably transfected with the human cannabinoid subtype 2 receptor (CNR2; hCB2) [28 (link)] or the human mu-opiod receptor (MOR, CHO-hMOR) [29 (link)]. CHO cells stably expressing hCB1 receptors (CNR1; CHO-hCB1) were purchased from DiscoverRx Corporation (Fremont, CA). CHO-hCB2 and CHO-hMOR cell lines were cultured in DMEM (Mediatech Inc., Manassas, VA) while CHO-hCB1 cells were cultured in HAM’s F-12 K media (ATCC, Manssas, VA). Media for all cell types contained 10% fetal calf serum (Gemini Bioproducts, Sacramento, CA), 0.05 IU/mL penicillin, 50 μg/mL streptomycin (Invitrogen, Carlsbad, CA), and 250 μg/mL of Geneticin (or G418; Sigma-Aldrich, St. Louis, MO). All cell types were maintained in a humidified chamber at 37°C with 5% CO₂, harvested when flasks reached approximately 80% confluency, and only cells from passages 1–15 were used in all experiments.

All experiments were conducted using intact cells or membranes prepared from Chinese hamster ovary (CHO) cells stably transfected with either human cannabinoid type-1 receptors (CHO-hCB1), human cannabinoid type-2 receptors (CHO-hCB2) (Shoemaker et al., 2005 (link)), or human mu opioid receptors (CHO-hMOR) (Seely et al., 2012 (link)). CHO-hCB1 cells were purchased from DiscoverRx Corporation (Fremont, CA, USA) and cultured in HAM’s F-12 K media (ATCC, Manassas, VA, USA). CHO-hCB2 and CHO-hMOR cells were cultured in DMEM (Mediatech Inc., Manassas, VA, USA). All media contained 10% Fetalplex, (Gemini Bioproducts, Sacramento, CA, USA), 0.05 IU/mL penicillin, 50 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), and 250 μg/mL of Geneticin (G418; Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in a 37°C humidified incubator with 5% CO₂ and harvested with PBS (10 mM)/EDTA (1 mM) when 80% confluent. All cells used were maintained between passages 4–15.