iHPCs were collected using cold (4°C) sterile filtered and degassed FACS buffer (1X DPBS, 2% BSA, and 0.05mM EDTA) spiked with human SCF (5 ng/ml). Cells were then filtered through 70 μm mesh to remove large clumps, washed with spiked FACS buffer (300 × g for 5 min 18°C), then stained (1:200) using spiked FACS buffer on ice for 1 hour in the dark using the following antibodies: anti CD34-FITC clone 561, anti CD41-PE clone HiP8, anti CD43-APC clone CD43-10G7, anti CD45-APC/Cy7 clone HI30 (Tonbo Biosciences), anti CD235a-PE/Cy7 clone HI264, and ZombieViolet™ live/dead stain, all from BioLegend unless noted. After staining, iHPCs were washed once with spiked FACS buffer and suspended using spiked FACS buffer (500–700 μl) and sorted utilizing the BD FACSARIA Fusion (BD Biosciences). Sorted cells were collected in cold basal iHPC differentiation medium spiked with SCF (10 ng/ml). Collected CD43+ iHPCs were then plated for iMGL differentiation as mentioned above. iMGLs were suspended in FACS buffer and incubated with human Fc block (BD Bioscience) for 15 min at 4°C. For detection of microglial surface markers, cells were
