The KOLF2 human iPSC line was maintained in mTeSR-E8 medium (STEMCELL Technologies, 05990) on recombinant human vitronectin (rhVTN-N)–coated plates (Gibco, A14700). For macrophage differentiation, pluripotent colonies were lifted using ReLeSR (STEMCELL Technologies, 05872) and plated onto irradiated MEFs on gelatin-coated 100-mm tissue culture plates in Advanced Dulbecco’s minimum essential medium (DMEM)–F12 (Thermo Fisher, 12634028), 20% knockout serum replacement (Gibco, 10828028), 1% l-glutamine, 1% penicillin/streptomycin, and 2-mercaptoethanol (7 μl/liter) supplemented with recombinant human fibroblast growth factor (rhFGF) basic (4 ng/ml) (R&D Systems, 233-FB-025). Once colonies were large but not touching each other, they were lifted using collagenase type IV (1 mg/ml) (Gibco, 17104-019) and dispase II (1 U/ml) (Gibco, 17105-041) diluted in a 1:1 ratio in Advanced DMEM-F12. Detached colonies were gently added to a 15-ml tube using a 10-ml pipette to avoid breaking them up and were centrifuged at 300g for 3 min. After removing the supernatant, the colonies were gently resuspended in fresh Advanced DMEM-F12 to be washed and recentrifuged. This wash step was carried out a total of three times. Colonies were then transferred to nonadherent 100-mm plates in 13 ml of Advanced DMEM-F12 (Thermo Fisher, 12634028), 20% knockout serum replacement (Gibco, 10828028), 1% l-glutamine, 1% penicillin/streptomycin, and 2-mercaptoethanol (7 μl/liter) without FGF. After 4 days, embryoid bodies had formed and these were collected in suspension, centrifuged, and then plated in gelatin-coated plates in X-VIVO 15 media (Lonza, BE02-060Q), 1% l-glutamine, 1% penicillin/streptomycin, and 2-mercaptoethanol (7 μl/liter) supplemented with human M-CSF (50 ng/ml) and IL-3 (0.5 mg/ml). Medium was changed twice weekly. After 20 to 30 days, macrophage precursors began to appear in the media and were collected in suspension, strained using a 40-μm cell strainer, and plated on standard tissue culture plates in RPMI 1640, 10% FBS, and 1% l-glutamine supplemented with M-CSF (100 ng/ml). After 7 days, mature macrophages were used for assays.

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