F4 80 pe

Primary bone marrow derived macrophages (BMDM) were recovered from bone marrow suspensions of tibia and femur of LDLR^−/−LMP7^−/− and LDLR^−/− littermate mice as described previously³¹ . For differentiation in macrophages, cells were incubated in RPMI-1640 (Gibco Life Technologies) containing 10% fetal calf serum, 1% penicillin/streptomycin, and 10% of L929-conditioned RPMI (as a source of Macrophage Colony Stimulating Factor, M-CSF) for 7 days. Macrophage differentiation was verified by flow cytometry via staining for CD45 (APC, BioLegend), F4/80 (PE, Abcam) and C11b (V450, BD Horizon) on day 7. Mouse ear fibroblasts have been isolated and cultivated as described previously^{32 (link)}.
For individual experiments, BMDM or fibroblasts were treated with 100 U/ml IFN-γ (recombinant, Biomol) for varying times or with medium containing varying concentrations of hydrogen peroxide (H₂O₂; Sigma) at 37 °C for 60 minutes. For survival analyses, H₂O₂-treated BMDM were harvested by scraping, and counted in a hemocytometer. Numbers of survived cells were expressed as percentage of cell count of the corresponding untreated control. For polarization of macrophages into the M1 and M2 state, BMDMs were incubated with 100 U/ml IFN-γ or 5 U/ml IL-4 (Promokine) at 37 °C for 6 hours, respectively.

The SVF was isolated from inguinal subcutaneous or epidydimal visceral fat depot of male FGF21KO and WT mice after rmFGF21 treatment or fat transplantation. The mice were anesthetized and the fat pads were removed and digested in 0.1% (w/v) collagenase type I (Invitrogen) for 30 min at 37 °C with gentle shaking. The digestion mixture was passed through a 100 mm cell strainer (BD Biosciences) and centrifuged at 800×g for 10 min at 4 °C. The SVF pellets were collected and washed twice, 1 × 10⁵ freshly isolated cells were triple stained with F4/80-PE (Abcam, ab105156, clone CI:A3-1, 1:50), cd206-Alexa Fluor 647 (Biolegend, 141712, clone C068C2, 1:100) and cd11c-FITC (BD Biosciences, BD 557400, clone HL3, 1:100) on ice for 30 min in dark. Species-matched IgG was used as non-specific isotype controls. After washing, the cells were analyzed with LSR Fortessa Cell Analyzer (BD Biosciences).