*2.3. Peritoneal Macrophages*

Peritoneal exudate cells were induced in mice by an intraperitoneal injection of 0.5 mL of 3% thioglycollate (BD DIFCO, Franklin Lakes, NJ, USA). After 4 days, mice were anesthetized with ketamine and xylazine, and then killed by cervical dislocation. Peritoneal exudate cells were washed from the peritoneal cavity of mice by lavage with 5 mL of icecold, sterile phosphate buffered saline (PBS). Cells were washed with PBS and re-suspended in Dulbecco's modified Eagle medium (DMEM) (Sartorius, Israel) supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin, and 1% L-glutamine (Biological industries/Sartorius, Beit Haemek, Israel). Cell viability was determined by MTT colorimetric assay in which a yellow tetrazole, is reduced to purple formazan in living cells (MP Biomedicals, LLC, Solon, OH, USA). The resultant color was measured at 450 nm using a Biotek PowerWave XS Microplate Reader.

#### *2.4. Nitric Oxide (NO*•*) Determination*

Peritoneal macrophages were seeded at a density of 2.5 × 10<sup>5</sup> cells/well in 96-well plates and incubated overnight at 37 ◦C and 5% CO2. On the following day, the medium was changed to fresh DMEM containing 5 μg/mL CBD, THC, or cannabis extracts. The cells were then stimulated for 24 h by the addition of lipopolysaccharide (LPS) to a concentration of 1 μg/mL. After 24 h, cell supernatants (SNs) were harvested for nitric oxide radical (NO•) assay by addition of 100 μL SN to an equal volume of Griess reagen<sup>t</sup> (1% sulfanilamide, 0.1% naphthalene diamine, and 2% H3PO4). After 10 min of incubation, the resultant color was measured at 550 nm. The amount of NO• produced, and any inhibition by the tested materials, was calculated from a standard curve prepared with NaNO2. Controls: nonactivated cells, activated cells + vehicle, activated cells + 1400W dihydrochloride (NOS2 inhibitor, Enzo Life Sciences Inc., Lausen, Switzerland).
