Bovine serum albumin (bsa)

Bone marrow-derived monocytes were isolated from C57BL/6J mice 4–8 weeks of age using an established procedure³² . Briefly, bone marrow cells were flushed from extracted tibiae and femurs using harvest solution (PBS with 0.02% Bovine Serum Albumin (STEMCELL), 1 U ml⁻¹ Heparin (STEMCELL), 300 U ml⁻¹ DNase (Worthington)). Homogenised bone marrow suspension was strained through 40-µm mesh and plated on non-tissue culture-treated 15 cm petri dishes. Macrophages were allowed to differentiate for a period of 6 days in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco), 1% penicillin-streptomycin (Gibco), and 40 ng ml⁻¹ of recombinant murine CSF1 (Biolegend) before passaging. Macrophages were seeded at density of 1.5 × 10⁶ per well of a six-well plate for all experiments.
Microglial cells were isolated from P2 C57BL/6J mouse pups using a previously established procedure^{33 (link)}. Briefly, murine brains were dissected out and meninges were removed under a microscope. Brains were dissociated using 0.25% trypsin with DNase and the resulting single-cell suspension was strained through 40-µm strainers. Cells were seeded in poly-d-lysine-coated T75 flasks, grown in the same medium as BMDMs, and cultured for 10 days, and then collected by vigorous shaking. Microglial cells were seeded at a density of 8 × 10⁵ per well of a six-well plate for all experiments.

10% BSA solution was prepared by dissolving bovine serum albumin in NSA medium (Stem Cell Technologies) without supplements. Next, this solution was incubated in a water bath at 37°C until completely dissolved and then sterile-filtered using a 0.2 micron filter. In the meantime, 100mM stocks of fatty acids were prepared in 50% ethanol (sodium palmitate) or 100% ethanol (all other fatty acids). Subsequently, these were diluted in 10% BSA to obtain 2mM sodium palmitate and 1mM palmitoleate and sapienate. As control, 100% ethanol was diluted 1:50 in 10% BSA and processed the same way as fatty acids. Next, they were incubated in water bath at 37°C for at least 1 hour to allow conjugation. Conjugated fatty acids were aliquoted to avoid freeze-thaw cycles and stored at -20°C.