Wang Wei, Qin Yi, Wang Yaru, Zou Jiejie, Chen Jing, Chen Jinwu, Zhang Yan, Geng Ming, Xu Zhongdong, Dai Min, Pan Lilong
School of Life Sciences, Hefei Normal University, Hefei 230601, Anhui, China.
School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, Anhui, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Jul 25;36(7):1431-1439. doi: 10.13345/j.cjb.190517.
The purpose of this study is to provide a culture for mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) and to characterize their molecular and cellular biology. The cell number and purity from the primary culture were assessed by cell counter and flow cytometry, respectively. Morphological features were evaluated by inverted microscope. Phagocytosis by macrophages was detected by the neutral red dye uptake assay. Phenotypic markers were analyzed by real-time fluorescent quantitative PCR. Our results show that the cell number was much higher from culture of BMDM than PM, while there was no significant difference regarding the percentage of F4/80+CD11b+ cells (98.30%±0.53% vs. 94.83%±1.42%; P>0.05). The proliferation rate of BMDM was significantly higher than PM in the presence of L929 cell conditioned medium, by using CCK-8 assay. However, PM appeared to adhere to the flask wall and extend earlier than BMDM. The phagocytosis capability of un-stimulated BMDM was significantly higher than PM, as well as lipopolysaccharide (LPS)-stimulated BMDM, except the BMDM stimulated by low dose LPS (0.1 μg/mL). Furthermore, Tnfα expression was significantly higher in un-stimulated BMDM than PM, while Arg1 and Ym1 mRNA expression were significantly lower than PM. The expression difference was persistent if stimulated by LPS+IFN-γ or IL-4. Our data indicate that bone marrow can get larger amounts of macrophages than peritoneal cavity. However, it should be aware that the molecular and cellular characteristics were different between these two culture systems.
本研究的目的是为小鼠骨髓来源的巨噬细胞（BMDM）和腹腔巨噬细胞（PM）提供一种培养方法，并对其分子和细胞生物学特性进行表征。通过细胞计数仪和流式细胞术分别评估原代培养物中的细胞数量和纯度。通过倒置显微镜评估形态学特征。通过中性红染料摄取试验检测巨噬细胞的吞噬作用。通过实时荧光定量PCR分析表型标志物。我们的结果表明，BMDM培养物中的细胞数量比PM多得多，而F4/80+CD11b+细胞的百分比没有显著差异（98.30%±0.53%对94.83%±1.42%；P>0.05）。使用CCK-8试验，在L929细胞条件培养基存在下，BMDM的增殖率显著高于PM。然而，PM似乎比BMDM更早地附着在培养瓶壁上并伸展。未刺激的BMDM的吞噬能力显著高于PM，以及脂多糖（LPS）刺激的BMDM，但低剂量LPS（0.1μg/mL）刺激的BMDM除外。此外，未刺激的BMDM中Tnfα表达显著高于PM，而Arg1和Ym1 mRNA表达显著低于PM。如果用LPS+IFN-γ或IL-4刺激，表达差异仍然存在。我们的数据表明，骨髓比腹腔能获得更多的巨噬细胞。然而，应该注意的是，这两种培养系统之间的分子和细胞特征是不同的。
