Il 4 214 14

For mouse cells, whole spleens were extracted from donor (CD45.1) mice and were mechanically crushed in PBS to be filtered in a 70 μm Cell Strainer (Corning). Following Red Blood Cell lysis (420301, Biolegend), cells were plated at 3.0E6 cells/ml in 1640 RPMI (01-100-1A, Biological Industries) supplemented with 10% HI FBS (04-127-1A, Biological Industries), 50 μM β-Mercaptoethanol, P/S (03-031-1B, Biological Industries), 10 μg/ml LPS (sc-3535, SantaCruz Biotechnology) and 10 ng/ml IL4 (214-14, Peprotech) was added immediately after extraction.
Cells were cultured 16–24 h and washed in PBS before transfections and plated in the same activation medium without P/S for 8–16 h following electroporations. Parameters were 1350 v 30 ms 1 pulse at 4.0E5 cells/μl in buffer R for 10 μl tips. For RNP: Cas9 (1081059, IDT) and gRNA (IDT) complexes assemblies were generated 20 min prior of transfection with 18.3 pmol Cas9 and 38 pmol gRNA per 1E06 cells. Transductions were performed no later than 5 min following electroporation with a 50,000 MOI of rAAV-DJ and cells were analyzed 2 days following electroporation. For co-culture with CD40LB feeders, splenic lymphocytes were seeded as previously described^{17 (link)}.

Bone marrow was flushed from mouse femurs and tibias and dissociated into a single-cell suspension. After red blood cell depletion, cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin solution. To induce BMDM differentiation, 10 ng/mL macrophage colony-stimulating factor (M-CSF) (416-ML, R&D Systems) or 15% L929-conditioned media were added every 2 days for 7 days [24 (link)]. BMDMs were stimulated with 100 ng/mL LPS (L4391, Sigma) and 20 ng/mL IFN-γ (315-05, PeproTech) to induce M1 polarization, or 10 ng/mL IL-4 (214-14, PeproTech) and 10 ng/mL IL-13 (413-ML, R&D Systems) to induce M2 polarization. For MMe activation, BMDMs or RAW264.7 cells were treated with 0.4 mM palmitate, 10 nM insulin, and 30 mM glucose for 24 h [25 (link)].