Roswell park memorial institute (rpmi)

PBMCs of healthy individuals and UC patients were isolated. The cell pellet was resuspended in RPMI (Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 1×10⁶ cells/ml. Additionally, 500 µl RPMI with 10% FCS and 1% penicillin-streptomycin (Sigma-Aldrich, St Louis, MO, USA) were added to each well and sample. Wells containing PBMCs and RPMI or PBMCs, RPMI and anti-CD3 (1 µg/ml, BioLegend, San Diego, CA, USA) served as negative and positive controls, respectively. ritonavir was dissolved in 100% ethanol (10 mg/ml). For analysis of the effect of ritonavir, 100 µg/ml ritonavir (Sigma-Aldrich, Deisenhofen, Germany) was added. Cells were incubated for 72 h. The content of each well was centrifuged at 1400 g for 5 min. The pellet was resuspended in FACS buffer and labeled for flow cytometry.
To differentiate between live and dead cells, staining with Zombie Green™ Fixable Viability Kit (BioLegend) was performed according to the manufacturer's instructions.

BMDCs were cultured as described (69 (link)). Briefly, the femurs and tibiae of UBC-tdTomato mice were rinsed with 70% ethanol and then with sterile PBS. The bone marrow was flushed with sterile RPMI (Gibco), pipetted several times to break down the clumps, and filtered through 70 μm Nylon mesh. Two million bone marrow cells were seeded in 10 cm Petri dishes (Alpha, Taiwan) in complete RPMI containing 10% fetal bovine serum, 1% Penicillin/Streptomycin, 1% L-Glutamine, 0.1% 2-Mercaptoethanol, and 20 ng/ml recombinant mouse GM-CSF (BioLegend) and cultured in a 37˚C incubator with 5% CO₂. Ten milliliter of fresh medium with 20 ng/ml mouse GM-CSF was added on day 3. Half of the medium was replaced with fresh GM-CSF-containing medium on days 6 and 8. The nonadherent and loosely adherent cells were harvested by gentle pipetting on day 10 and labeled as immature BMDC. Mature BMDCs (mBMDCs) were produced from the immature BMDCs by treatment with 200 ng/ml LPS (Invivogen) overnight. The nonadherent and poorly adherent cells were harvested and labeled as mBMDC

Murine bone marrow macrophages were isolated from femur and tibia of 8-week old C57BL/6N mice. Isolated monocytes were differentiated to macrophages in RPMI supplemented with 20 ng/ml mMCSF (macrophage stimulating colony factor; BioLegend), 5% FCS and 1% penicillin/streptavidin (Thermo Fisher Scientific) for 7 days. For analysis macrophages were cultured in RPMI (Gibco, Invitrogen Corp.) supplemented with 5% FCS, 5 ng/ml mMCSF and were seeded at a density of 2 × 10⁴ cells/96-well and 1 × 10⁵ cells/6-well for further experiments. For polarization into M1—and M2 phenotype macrophages were incubated for 4 h with LPS (Lipopolysaccharide) and IFNy (Interferon gamma) (each 10 ng/ml; Peprotech) or IL-4 (10 ng/ml; Peprotech). Afterwards 0.5 µM Regorafenib was added into the culture media for additional 24 h.