12 well cell cluster plates

Liver nonparenchymal cells (NPC) were isolated using a liver dissociation kit and the gentle MACS dissociator (both Miltenyi Biotec) as recommended by the manufacturer with some modifications. In brief, after liver dissociation, cells were resuspended in 1 mL cold HBSS buffer (w/o Ca²⁺ and Mg²⁺). The cell suspension was mixed with a double volume of freshly prepared 30% HistoDenZ (Sigma-Aldrich) and overlaid with 1 mL of cold HBSS buffer. After centrifugation (1500× g, 4 °C, w/o break), the cell containing interphase was retrieved and washed. Erythrocytes were lysed using a hypotonic buffer.
Spleen cells were isolated using a 40 µM cell strainer (Greiner Bio-One) to obtain a single-cell suspension. In parallel settings, spleen cells were resuspended in medium (IMDM, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin (all from Sigma-Aldrich, Deisenhofen, Germany), and 50 µM ß-mercaptoethanol (Roth, Karlsruhe, Germany)) containing 5% FCS (PAN).
Bone marrow cells (2 × 10⁵/mL) were seeded in 12-well cell cluster plates (Greiner Bio-One) in an IMDM-based culture medium (see above) supplemented with 10 ng/mL recombinant murine GM-CSF (R&D Systems, Wiesbaden, Germany). Culture media was replenished on days 3 and 6 of culture.

Bone marrow cells (4 × 10⁵/mL) were seeded in 12-well cell cluster plates (1 mL) (Greiner Bio-One, Kremsmünster, Austria) in IMDM-based culture medium containing 5% FCS (PAN-Biotech, Aidenbach, Germany), 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM ß-mercaptoethanol (all from Sigma-Aldrich, Deisenhofen, Germany), and supplemented with 10 ng/mL recombinant murine M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany). Culture media was replenished every 3 days of culture. BMDM were subjected to experiments on days 7–9 of culture unless indicated otherwise.