24 well culturing plates

BMDMs were detached from the flask using Trypsin (Sigma). Cells were washed and re-seeded in 24-well culturing plates (Sarstedt, Nümbrecht, Germany) at a density of 2.5 × 10⁵ cells/well for cytokine and nitrite measurements, or at 0.5 million cells/well for gene expression analysis, in DMEM containing 1% FBS and 10,000 I.U./mL PenStrep, and rested overnight. In order to generate cells with an M1 phenotype, 100 ng/mL LPS-EK (InvivoGen, Toulouse, France) and 20 ng/mL IFN-γ (R&D Systems) were added. In order to generate cells with an M2 phenotype, 20 ng/mL each of, IL-4, IL-10, and TGF-β, were added (R&D Systems). frHMGB1 or dsHMGB1 were added at the indicated concentrations (1–9 μg/mL). PBS was included in all experiments as a negative control.

The BMDMs were detached from the flask using Trypsin-EDTA (Sigma, St. Louis, MO, USA). The cells were seeded in 24-well culturing plates (Sarstedt, Nümbrecht, Germany) at a density 5 × 10⁵ cells/well in DMEM containing 1% FBS and 10,000 I.U./mL PenStrep and rested overnight. For RNA-Seq, BMDMs from five mice were pooled before seeding; for qPCR verification, BMDMs from each individual mouse were seeded separately.
To generate cells with an M1 phenotype, 100 ng/mL of LPS-EK Ultrapure (InvivoGen, Toulouse, France) and 20 ng/mL of IFN-γ (R&D Systems, Minneapolis, MN, USA) were added. frHMGB1 and dsHMGB1 were used at a concentration of 5 µg/mL. LPS-EK Ultrapure (InvivoGen, Toulouse, France) was used at a concentration of 1 µg/mL. PBS was included in all experiments as a control. The BMDMs were stimulated 24 h at 37 °C with 5% CO₂ before RNA isolation.