A streamlined and comprehensive protocol for the generation and multi-omic analysis of human monocyte-derived macrophages

Macrophages are versatile immune cells with phenotypes ranging from inflammatory (M1-like) to anti-inflammatory (M2-like), shaped by diverse differentiation and polarization conditions. We present a standardized protocol to generate and characterize human macrophages from frozen PBMC-derived monocytes using cytokine profiling, transcriptomics, and snRNA-seq, including depolarization studies. This streamlined approach facilitates reproducible macrophage studies from biobanked samples, advancing both research and therapeutic applications. Overall design: Monocytes from 3 female donors were isolated from frozen peripheral blood mononuclear cells and differentiated into macrophages over 6 days using 25ng/mL M-CSF in RPMI media with 10% FBS and 1% Penicillin-Streptomycin. On day 7 macrophages were activated into M1-like cells using 10ng/mL LPS and 20ng/mL IFNgamma.