Abstract: BACKGROUND: Macrophage polarization demonstrates significant potential in disease treatment, particularly in areas such as cancer, inflammation, and autoimmune diseases. Establishing standardized in vitro models can lay the groundwork for in-depth research into the mechanisms of macrophage polarization.
OBJECTIVE: To observe the in vitro growth characteristics of bone marrow-derived macrophages from C57BL/6 mice and to establish a standardized in vitro model for M1 and M2 macrophage polarization.
METHODS: Femurs and tibias of C57BL/6 mice were aseptically separated, and the contents of the bone marrow cavity were collected. After filtering through a mesh and lysing erythrocytes, the contents were resuspended in high-glucose DMEM containing 20 ng/mL macrophage colony-stimulating factor and inoculated in 6-well plates according to experimental requirements. On day 7, they were differentiated into mature mouse bone marrow-derived macrophages (M0 type). Then, 100 ng/mL lipopolysaccharide was used to induce polarization of M1 macrophages, and 20 ng/mL interleukin-4 was used to induce polarization of M2 macrophages. Flow cytometry and RT-qPCR were used to detect the expression of corresponding markers of macrophages in different polarization states. Western blot assay was used to detect the expression of M1 macrophage marker pathway proteins p-STAT1 and STAT1 and M2 macrophage marker pathway proteins p-STAT6 and STAT6.
RESULTS AND CONCLUSION: (1) After stimulation with 20 ng/mL macrophage colony-stimulating factor for 7 days, the positive staining rate of macrophage surface marker F4/80 by flow cytometry reached 98.1%. (2) After bone marrow-derived macrophages were stimulated with 100 ng/mL lipopolysaccharide for 6 hours, the positive staining rates of F4/80 and CD86 were approximately 35%, and the mRNA expressions of M1 macrophage markers inducible nitric oxide synthase, interleukin-6, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 by RT-qPCR were significantly higher than those in the control group (P < 0.01). (3) After bone marrow-derived macrophages were stimulated with 20 ng/mL interleukin-4 for 24 hours, the mean fluorescence intensity of CD206 was significantly increased, and the mRNA expressions of M2 macrophage markers Chi3l3 (Ym1), interleukin-10, and arginase-1 by RT-qPCR were significantly higher than those in the control group (P < 0.01). (4) Western blot assay results showed that lipopolysaccharide-induced M1 macrophages' signature pathway p-STAT1 was significantly activated; interleukin-4-induced M2 macrophages' landmark pathway p-STAT6 was significantly activated. The above results indicate that lipopolysaccharide and interleukin-4 effectively induce bone marrow-derived macrophages to polarize into M1 and M2 macrophages, respectively.

Key words: bone marrow-derived macrophage, BMDMs, macrophage polarization, lipopolysaccharide, interleukin-4, M1 macrophage, M2 macrophage