Effect of recombinant human Annexin A5 on gene expression of bone marrow-derived macrophages differentiating into osteoclasts
Primary BMMs were obtained from male C57BL/6 miceaged 4-6 weeks. The mice were euthanized, and femurs and tibias were removed. BMMs 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 osteoclast differeation, 100000 BMDMs were seeded in 12 well plates. After 24 hours of cell seeding, the regular medium was replaced with culture medium (30ng/ml m-csf, control group, A-1, A-2,A-3 and A-4), culture medium containing  50 ng/mL RankL (model group, B-1, B-2, B-3 amd B-4), culture medium containing  50 ng/mL RankL and 2 ug/mL of Annexin A5 (Treat group, C-1, C-2,C-3 and C-4) for 5 days. Overall design: Total RNA was extracted using Trizol from BMMs after m-CSF and RANKL treatment with or without Annexin A5 (n = 4) for 5 days.