Dulbecco modified eagle medium (dmem)

Cell invasion was determined using 24-well transwells (8-μm pore size; Corning, New York, USA) precoated with Matrigel (BD Biosciences). A total of 1×10
⁵ cells were suspended in 100 μL Dulbecco’s modified Eagle medium (DMEM; BD Biosciences) with 1% fetal bovine serum and were added to the upper chamber, and 600 μL DMEM with 10% fetal bovine serum was added in the lower chamber. After 48 h of incubation, the cells remaining in the upper chamber were removed using cotton swabs. Cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with Giemsa. Cells in five microscopic fields were photographed at 200× magnification and counted.

Immune cells were isolated from colon tissues, peripheral blood, bone marrow and spleen by flow sorting. Single-cell suspensions from colon tissue dissection were prepared by manual mincing using scalpel followed by enzymatic digestion for 40 min at 37°C by Collagenase A 2.0 mg/mL (Roche, Canada) and DNase I 100U/mL (Roche, Canada) dissolved in DMEM (Gibco, USA) under continuous stirring. Digestion mixtures were resuspended by DMEM containing 10% FBS and then filtered through 70 μm nylon strainers (Falcon, BD biosciences, USA). Immune cells from peripheral blood,bone marrow and spleen were obtained by Ficoll-Hypaque (TBD science, China) density centrifugation 2000 rpm for 20 min.

Human skin fibroblasts were isolated from 3-mm skin punch biopsy (n = 15 for RTT and n = 15 for controls). Cells were cultured in DMEM (Sigma-Aldrich, Milan, Italy), containing 20% fetal calf serum [9] (link) (FCS, Sigma-Aldrich) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) (Lonza, Milan, Italy). Cells were incubated at 37 C° in a humidified atmosphere at 5% CO2 for 3 days. When fibroblasts growing from the dermal pieces formed a confluent layer, the dermal pieces were removed and trypsin/EDTA mixture (Sigma-Aldrich) was added to separate fibroblasts. Cells were transferred to 25-cm² culture flasks (Falcon, Perugia, Italy) and subcultured in 10% FCS/DMEM. Fibroblasts from passage 3 to 5 were used for the experiments. 1×10⁶ cells were seeded in each flask (25 cm²), whereas the experiments were performed when cells reached 70/80% confluence.

HSCs were isolated from WT and Cygb^−/− mice using the pronase-collagenase digestion method as described previously66 (link) and were cultured on uncoated-plastic dishes (BD Falcon, Franklin Lake, NY, USA) or glass chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37 °C in a 5% CO₂/95% room air. Mouse hepatoma Hepa 1–6 cells (CRL-1830) obtained from American Type Culture Collection (Manassas, VA, USA) were maintained on uncoated-plastic culture plates (BD Falcon) in DMEM supplemented with 10% FBS and antibiotic.

Tumor and adjacent tissues were cut into pieces (< 1 mm³) in DMEM (Gibco, Germany) with 10% FBS (Gibco) and digested using a MACS tumor dissociation kit (Miltenyi Biotec, Germany) for 30 min on a rotor at 37 °C. The digested mixture was filtered using a 70 µm cell strainer (BD Falcon, USA) in DMEM to obtain dissociated cells. Dissociated cells were centrifuged at 330 × g at 4 °C for 10 min. After removing the supernatant, cells were treated with red blood cell lysis buffer (Solarbio, China) for 15 min on ice. After washing twice with PBS, cells were resuspended in sorting buffer (PBS supplemented with 2% FBS). Finally, cells in 10 μL suspension were counted using an inverted microscope and a hemocytometer. Cell viability was assessed via 0.1% trypan blue staining.

To generate BMDM, the bone marrow cells from femurs and tibias from mice were harvested and cultured as previously described [5 (link),28 (link)]. Briefly, isolated cells were incubated in Dulbecco’s Modified Eagle Media (DMEM, Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Grand Island, NY)), 1% penicillin/streptomycin, 1% glutamine, and 20% L929 cell supernatant (containing macrophage colony stimulating factor). On day 7 in culture the cells were washed, counted and replated in DMEM media (without L929 supernatant) at a density of 6-8x10⁶ cells/well (6-well plate, Falcon polystyrene). Cells were classically activated (M1 condition) with LPS (100 ng/ml, Sigma-Aldrich L2880) + IFN-γ (20ng/mL, eBioscience, San Diego, CA), alternatively activated (M2 condition) with IL-4 (20ng/mL, eBioscience) or received media alone (M0 condition). The LPS+ IFN-γ condition is used to simulate infectious and/or autoimmune conditions in which Th1 cells produce IFN-γ while pathogens or tissue damage provide PAMPs or DAMPs, respectively. Cells were harvested at the indicated time points, generally 24 hours post-stimulation, by washing in phosphate buffered saline (PBS) before cell lysis in miRVana Lysis buffer (Life Technologies) for total RNA isolation.