*2.5. Flow Cytometry*

5 × 10<sup>5</sup> cells/sample were washed once in ice-cold staining buffer (PBS containing 1% FBS, pH 7.2). Then, cells were stained in the dark at 4 ◦C for 30 min with fluorochromelabeled anti-mouse mAb (Biolegend, San Diego, CA, USA), specific for cell surface antigens: F4/80, I-Ad (MHC class II), and CD16/32. Cells were subsequently washed, re-suspended in staining buffer, and analyzed by flow cytometry.

#### *2.6. RNA Extraction and Real-Time PCR Analysis*

Total cellular RNA was extracted using RNeasy Mini Kit columns (Geneaid, New Taipei City, Taiwan), according to the manufacturer's protocol. One microgram of total RNA was used to synthesize cDNA using a high-capacity cDNA kit (Applied Biosystems, Waltham, MA, USA), following the supplier's instructions. Detection of transcript levels of CB1 and CB2 was performed using a TaqMan Gene Expression Assay Kit (Applied Biosystems, Waltham, MA, USA), with HPRT-1 as a reference. All primers were purchased from Applied Biosystems (Waltham, MA, USA). Real-Time PCR reactions were conducted using a QuantStudio 5 instrument (Applied Biosystems, Waltham, MA, USA). Data were analyzed using the QuantStudio design and analysis Software (Applied Biosystems, Waltham, MA, USA).

#### *2.7. Induction of Colitis in Mice*

Colitis was induced in C57BL/6 mice with 2% DSS dissolved in drinking water given ad libitum (Days 1–7), and then replaced with plain drinking water for 3 days. Then, 5 mg/kg cannabis/cannabinoids were prepared in 5% Cremophor EL (Sigma, St. Louis, MO, USA), 5% ethanol (Gadot, Haifa, Israel) in PBS, and 0.1 mL were administered IP every other day, starting from Day 1. Body weight and stool were monitored once a day. Changes of body weight are indicated as loss of baseline body weight (% of initial weight). Clinical score (0–9) included: stool score (0–3), rectal score (0–3), and general clinical parameters (fur texture, behavior, and posture, 0–3). On the tenth day of colitis induction, blood was collected from the mouse tails into ethylenediaminetetraacetic acid (EDTA)-coated capillary tubes, and then the mice were anesthetized using ketamine and xylazine, and then killed by cervical dislocation. The intestines were excised, measured, and carefully rinsed with saline. Blood tubes were centrifuged at 1500 rpm, room temperature, for 5 min; plasma was collected and kept at −80 ◦C for cytokine and chemokine analysis.

#### *2.8. Histopathology and Immunohistochemistry*

Colon tissue was fixed in 4% buffered formaldehyde (Bio-Lab, Jerusalem, Israel) and embedded in paraffin. For histology, the sections were stained with H&E according to standard protocols. Histological scoring (0–9) was based on 3 parameters: Crypt damage (0–3), percent involvement (0–3), and damage to bowel wall structure (0–3).

For immunostaining, paraffin embedded sections were heated to 60 ◦C, deparaffinized using xylene, dehydrated using ethanol, and washed with H2O. Sections were treated with 3% H2O2 and antigens retrieved by incubation with 1 mg/mL pronase. Then, the samples were washed in PBS and blocked in CAS blocking reagen<sup>t</sup> (Rhenium, Modi'in, Israel). The slides were stained with anti-F480 (Bio-Rad, Hercules, CA, USA). Anti-rat IgG universal immune peroxidase polymer (Nichirei Biosciences Inc., Tokyo, Japan) was used as secondary antibody. Sections were incubated with Stable Peroxidase Substrate Buffer (Thermo Scientific, Waltham, MA, USA), washed with H2O, and analyzed on a BX41 microscope (Olympus Corporation, Tokyo, Japan).

#### *2.9. Proinflammatory Chemokine and Cytokine Analysis*

Peritoneal macrophages were activated for 24 h with LPS, in the presence of 5 μg/mL CBD, THC, or cannabis extracts. The supernatant was collected and analyzed using a LEGENDplex ™ MU Macrophage/Microglia Panel cytokine array assay (Biolegend, San Diego, CA, USA), according to the manufacturer's instructions.

Plasma samples from DSS model mice were analyzed using a LEGENDplex ™ Mouse Inflammation Panel cytokine array (Biolegend, San Diego, CA, USA), according to the manufacturer's instructions.
