Effect of human recombinent Annexin A5 on gene expression of bone marrow derived macrophages polarizaionn with LPS and IFN-gama
Primary BMDMs were obtained from male C57BL/6 mice aged 4-6 weeks. The mice were euthanized, and femurs and tibias were removed. BMDMs were flushed out of the bones using a cold culture medium, filtered through a 70-um cell strainer , and centrifuged at 1500 rpm for 5 min. The cells were suspended in cold culture medium supplemented with 20 ng/ml M-CSF and cultured for five days, with half of the medium being replaced on day three and full replacement on day 5 before being used for subsequent experiments.The culture medium was alpha-MEM supplemented with 10% FBS and 1% penicillin-streptomycin-gentamicin solution. Mature BMDMs were double-stained using flow cytometry antibodies CD16/32 and F4/80 for identification. The recombinant protein AnxA5 used in this study was produced by SEME Cell Technology Co., Ltd, China. To investigate the role of AnxA5 in regulating macrophage polarization in an inflammatory environment, 100000 BMDMs were seeded in 12 well plates. After 24 hours of cell seeding, the regular medium was replaced with culture medium (control group, 1-1, 1-2 and 1-3), culture medium containing 1 ug/mL lipopolysaccharide and 30 ng/mL interferon gama (model group, 2-1, 2-2 and 2-3), culture medium containing 1 ug/mL LPS, 30 ng/mL IFN gama, and different 2 ug/mL of AnxA5 (Treat group, 4-1, 4-2 and 4-3) for 6 hours Overall design: Total RNA was extracted using Trizol from BMDMs after LPS and IFN-gamma treatment with or without AnxA5 (n = 3) for six hours.