Isolation,cultivation,identification,and induction of M1/M2 polarization in bone marrow-derived macrophages from C57BL/6 mice

Tan Yuhang; Li Bo; Tang Minghong; Sun Zeyu; Luo Xu

doi:10.12307/2026.087

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.