A streamlined and comprehensive protocol for the generation and multi-omic analysis of human monocyte-derived macrophages [bulk RNA-Seq]

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: All samples are human monocyte derived macrophages cultured for 6 days in M-CSF at a density of 100,000 cells per well in a 96 well plate. On day 7 macrophages were activated with a cytokine cocktail to induce an M0, M1 or M2-like phenotype and then we characterized the transcriptional signature.