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目的建立一套简便易行、高效可靠的小鼠子宫巨噬细胞的分离纯化及鉴定方法。方法5只雌鼠处死并获取子宫,利用酶消化法和机械法相结合制备小鼠子宫组织单细胞悬液,密度梯度离心法收集子宫巨噬细胞,贴壁培养获得纯化的巨噬细胞。采用锥虫蓝拒染法鉴定巨噬细胞活率,利用鸡红细胞吞噬试验检测巨噬细胞吞噬活性,并通过酸性磷酸酶染色、非特异性酯酶染色和CD14免疫细胞化学染色鉴定巨噬细胞纯度。结果采用该法每只小鼠可获得子宫巨噬细胞3×105~5×105个,且活率达到(85.35±0.43)%,酶化学和免疫细胞化学鉴定阳性率分别达到了(86.12±1.19)%(、85.83±0.57)%和(84.02±1.64)%。结论成功建立了小鼠子宫巨噬细胞的分离纯化方法,并能获得较高的细胞存活率和纯度。
Objective To establish a simple, efficient and reliable method for the isolation and purification of mouse uterine macrophages. Methods Five female rats were sacrificed and the uterus was obtained. Uterine tissue suspension was prepared by enzymatic digestion and mechanical method. Uterine macrophages were collected by density gradient centrifugation and purified macrophages were obtained by adherent culture. Macrophage cell viability was assessed by trypan blue exclusion staining. The phagocytic activity of macrophages was determined by chicken erythrocyte phagocytosis assay. The purity of macrophages was identified by acid phosphatase staining, nonspecific esterase staining and CD14 immunocytochemical staining. Results According to this method, 3 × 105 ~ 5 × 105 uterine macrophages were obtained per mouse, and the viability reached 85.35 ± 0.43%. The positive rates of enzyme chemistry and immunocytochemistry were 86.12 ± 1.19 )% (, 85.83 ± 0.57)% and (84.02 ± 1.64)%, respectively. Conclusion The method of isolation and purification of murine uterine macrophages was successfully established and high cell viability and purity were obtained.