Comparative study of yield, purity, morphology, and LPS-induced cytokine responses of BMDMs and thioglycollate-elicited PEMs

Mouse bone marrow-derived macrophages (BMDM) and mouse peritoneal macrophages (PEMs) are the most commonly used primary cells in immunology studies. Direct and complete data for these models in the resting state are lacking. Cell yield, purity, and shape, and LPS-induced cytokine secretion profiles of BMDM and PEM under consistent experimental conditions. Femurs and tibias were removed from the same batch of C57BL/6 mice, and bone marrow was extracted. BMDMs were differentiated for 7 days with 20 ng/mL M-CSF. The mice were intraperitoneally given 3% sulfoglycolate medium, and PEMs were harvested 3 days later. Morphological changes of each type of cell were measured from day 0 until the mature state of resting. Cell yield was measured by hemocytometer and flow cytometry of F4/80+ CD11b+ double-positive cells. For functional comparison, mature macrophages were stimulated with LPS, and the secretion levels of IL-1β, IL-6, IL-12, IL-23, TNF-α, CCL5, CXCL9, and CXCL16 in culture supernatants were measured by ELISA at 6 h, 9 h, and 12 h post-stimulation. For their size and structure, mature BMDMs have an elongated spindle shape and connection. Mature PEMs are generally round or oval; some cells are polygonal after adhesion. The net yield ([2.41 ± 1.79] × 107/animal) and purity ([96.93 ± 0.53] %) were much higher than PEMs ([2.54 ± 0.15] × 106/animal, [71.18 ± 0.83] %), and they were statistically significant. The cell viability was higher than 93% for both cell types, with no difference between them. Upon LPS stimulation, PEMs secreted significantly higher levels of IL-1β and IL-6 at all measured time points and IL-23 (at 12 h) compared to BMDMs (P < 0.0001). BMDMs produced greater amounts of IL-12 (at 9 h and 12 h) and TNF-α (at all time points). For chemokines, CXCL16 was higher in PEMs at 9 h and in BMDMs at 12 h, while CXCL9 was elevated in BMDMs only at 12 h. CCL5 showed no significant difference between the two cell types at any time point. The rest of the BMDMs and PEMs differ very much in size, yield, and purity. They also exhibit distinct cytokine and chemokine response patterns upon LPS stimulation. In vitro modeling studies apply them in different ways. BMDMs are better suited for experiments with a large number of targets, and PEMs have shorter maturation cycles. The choice between the two models should consider both baseline characteristics and their distinct functional response profiles. Researchers should choose a cell model that best suits the experimental constraints.