Generation of bone marrow-derived macrophages and CD4+ T cells from mice and human iPSCs

To generate BMDM, bone marrow cells harvested from femurs and tibias of 6–10-week-old control or EROS knockout mice were grown in complete RPMI medium supplemented with 100 ng/mL of murine M-CSF (PeproTech) for 3 days. At day 3, 10 mL of the same medium was added to the culture and differentiated macrophages were collected at day 7 for mass spectrometry or Western blot analysis. To isolate mouse CD4 lymphocytes, mouse spleens were homogenised by manual disruption and subjected to positive selection using CD4 L3T4 magnetic beads (Miltenyi Biotec) according to the manufacturer’s protocol. Cells were counted and resuspended at a concentration of 2 × 10⁶ cells per mL in complete RPMI medium for subsequent analysis.
Control or EROS-deficient human iPS were generated by CRISPR Cas 9-targeted deletion of 46 base pairs in exon 5 of the CYBC1 sequence (Yeung et al., 2017 (link)). Human macrophages were obtained from the differentiation of the human control or EROS-deficient iPS line following previously established method (van Wilgenburg et al., 2013 (link); Thomas et al., 2017b (link)). Macrophages were cultured in complete RPMI in the presence of human M-CSF (PeproTech) and were used at day 7 post-differentiation.