ten Hagen T L, van Vianen W, Bakker-Woudenberg I A
Department of Clinical Microbiology and Antimicrobial Therapy, Erasmus University Rotterdam, Netherlands.
J Immunol Methods. 1996 Jun 14;193(1):81-91. doi: 10.1016/0022-1759(96)00045-2.
A method is described using counterflow centrifugation elutriation to isolate macrophages from murine liver and spleen. In this study three, size fractionated, macrophage populations were collected. Isolation resulted in a high yield of pure Kupffer cells (total of 10 x 10(6) /g liver) and enrichment of splenic macrophages to 20%. In addition to standard methods such as non-specific esterase staining, the isolated macrophages were also characterized by flow cytometry using specific monoclonal antibodies. In addition, a rapid flow cytometry method was introduced to determine the percentage of macrophages based on autofluorescence. A strong correlation was found between the percentages of macrophages found by non-specific esterase staining and autofluorescence. Functional tests revealed differences between the isolated macrophages in terms of tumor necrosis factor-alpha (TNF-alpha) production, oxygen metabolism and the production of nitric oxide. However, no significant differences in phagocytic activity was observed between the fractions. After two weeks of culture without the addition of antibiotics the cells still exhibited the above mentioned functions.
描述了一种使用逆流离心淘析从鼠肝和脾中分离巨噬细胞的方法。在本研究中，收集了三个按大小分级的巨噬细胞群体。分离得到了高产率的纯库普弗细胞（总计10×10⁶/g肝脏），并且脾巨噬细胞富集至20%。除了非特异性酯酶染色等标准方法外，还使用特异性单克隆抗体通过流式细胞术对分离的巨噬细胞进行表征。此外，引入了一种基于自发荧光的快速流式细胞术方法来测定巨噬细胞的百分比。发现通过非特异性酯酶染色和自发荧光检测到的巨噬细胞百分比之间存在很强的相关性。功能测试揭示了分离的巨噬细胞在肿瘤坏死因子-α（TNF-α）产生、氧代谢和一氧化氮产生方面存在差异。然而，各组分之间未观察到吞噬活性的显著差异。在不添加抗生素的情况下培养两周后，细胞仍表现出上述功能。
