Clone 30 f11

Mammary cells were obtained as performed in earlier studies [15] (link), [22] (link). In brief, inguinal mammary gland or interscapular BAT samples were dissociated by scissors and then incubated with 5% fetal bovine serum containing collagenase (300 IU/mL, Sigma) and hyaluronidase (100 IU/mL, Sigma) for 60 min at 37°C. Samples were then centrifuged at 500 g for 5 min, and the cell fractions were incubated with 0.25% trypsin-EGTA for 3 min, then resuspended in Dispase (5 mg/mL, Sigma) and DNaseI (50 IU/mL, Takara) for 5 min, and red blood cell lysis (0.64% NH₄Cl) for 3 min before filtration through a 40 μm cell mesh. Antibodies were incubated in PBS with 5% FBS for 20 min. The following primary antibodies were used: Percp-cy5.5 conjugated anti-CD24 (eBioscience, Clone M1/69), APC conjugated anti-CD29 (eBioscience, Clone HMB1-1), PE-cy7 conjugated anti-CD31 (eBioscience, Clone 390), PE-cy7 conjugated anti-CD45 (eBioscience, Clone 30-F11). The positive antibody signals were gated based on fluorescence minus one (FMO) control every time. Cell sorting was performed on FACSAria, and the data were read using Flowjo7.6.1 software.

Mice were injected intraperitoneally with methoxy‐XO4 (Tocris) at 10 mg/kg bodyweight in a DMSO/PBS mixture at 1:10 ratio as described previously with slight modifications (Heneka et al, 2013). Hippocampi were isolated 3 h after methoxy‐XO4 injection and processed into single‐cell suspension with a potter. The homogenate was filtered through a cell strainer (70 μm) and was separated by 37% Percoll gradient centrifugation at 800 g for 30 min at 4°C. The myeloid containing phase was collected and washed once with PBS. Fc receptor blocking antibody CD16/CD32 (1:200, clone 2.4G2, BD Bioscience) was applied in order to prevent unspecific binding, and dead cells were stained using the Fixable Viability Dye eFluor^® 780 (1:1,000, eBioscience) at 4°C for 20 min. Cells were washed once and then stained with primary antibodies directed against CD11b (1:200, clone M1/70, eBioscience), CD45 (1:200, clone 30‐F11, eBioscience), and CD36 (1:200, clone 72‐1, eBioscience) at 4°C for 20 min. Cells were washed again, and then, frequencies of viable methoxy‐XO4⁺ CD11b⁺CD45^low microglia cells were determined by flow cytometry using a FACS Canto II (BD Biosciences) and analyzed using FlowJo (Tree Star). WT mice injected with methoxy‐XO4 were used as controls to determine the methoxy‐XO4 threshold for non‐phagocytosing cells. Corresponding isotype control antibodies were used.