Analysis of activation markers, cytokine release, and phagocytosis in human monocyte-derived macrophages following RNP nucleofection. (A) Cell surface levels of indicated phenotypic markers measured by flow cytometry 5 d after nucleofection. (B) Efficiency of B2M-KO following nucleofection with two unique sgRNA:Cas9 RNPs. (C) Comparison of cell surface levels of CD80 and CD86 measured by flow cytometry 5 d after nucleofection. (D) ELISA measurements of TNF levels in cell culture medium of monocyte-derived macrophages following nucleofection. (E) Quantification of live monocyte-derived macrophages using imaging of live cell nuclei. Micrographs depict representative images of cultured cells. (F and G) Quantification of kinetics of particulate phagocytosis (F, myelin-pHrodo; G, beads-pHrodo). Micrographs depict representative images of phagocytosis following nucleofection, taken at the 5 h time point. Graphs in A–C depict MFI. Graphs in F and G depict intensity of pHrodo signal measured hourly following incubation with depicted particulate cargo. (H) Quantification of phagocytic index measured as AUC of data in G and H over 5 h of imaging. Data in A and B represent single measurements from two individual donors. Data in C–H are mean ± SD (n = 3) from three individual donors and a single experiment. Scale bars = 150 µm.