Recombinant m csf

Bone marrow cells were cultured in RPMI supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin/streptomycin and 2 mM L-glutamine. Macrophage differentiation was induced using 10 ng ml⁻¹ recombinant M-CSF (Preprotech) and terminal differentiation was confirmed by flow cytometric analysis after 8 days. Macrophages were exposed to 10 ng ml⁻¹ LPS (Sigma) for 2 h and stimulated for 6 h with palmitic acid (300 μM; Sigma) or cholesterol monohydrate crystals (200 μg ml⁻¹; Sigma) prepared as previously described³⁸ . Culture supernatant was centrifuged at 1,000g for 10 min at 4 °C and cytokine levels were measured by Eve Technologies using a Luminex-based mouse cytokine–chemokine magnetic bead panel.
Hepatic stellate cells, hepatic macrophages and purified lymphocytes were cultured in RPMI supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin–streptomycin, 2 mM L-glutamine, 1× non-essential amino acids and 0.05 mM 2-mercaptoethanol. LPS (10 ng ml⁻¹) and anti-CD3ε monoclonal antibody (145-2C11, 1 μg ml⁻¹) were used to supplement B and T lymphocyte cultures, respectively. Purified cells were grown in monocultures overnight before being directly co-cultured at 1:1 cell ratio. Hepatic macrophages were additionally grown in Transwell inserts. After 48 h, adherent cells were washed and collected for analysis.

Bone marrow derived macrophages (BMDMs) from the Csf1r^CreGCaMP5^fl reporter mice were isolated, cultured in 10% FBS 1% Pen/Strep-containing DMEM, and differentiated with addition of 10 ng/mL recombinant m-CSF (Peprotech) (every other day media changes) for a period of 7 days as previously described. 10 μg of immunogenic HT DNA (Invivogen) was complexed with a Lipofectamine transfection agent (ThermoFisher) in serum free-media and added to 1 million BMDMs in a 6-well multi-well plate with serum- and mCSF-containing media for each experiment.