利用流式细胞术检测LPS刺激活化后小鼠腹腔巨噬细胞体积的变化

Determination of volume change of LPS-stimulated mouse peritoneal macrophages by flow cytometry

目的探讨小鼠腹腔巨噬细胞经脂多糖(LPS)刺激活化后体积的变化。方法 LPS刺激小鼠腹腔巨噬细胞3和24 h后,用倒置显微镜观察细胞的形态,用ELISA法检测细胞上清中IL-6的含量,用流式细胞术定量检测细胞活化后的体积。结果小鼠腹腔巨噬细胞在正常状态下呈圆型,细胞边缘光滑无伪足,胞质内无空泡。经LPS刺激3 h后,小鼠腹腔巨噬细胞的表面积变大,伸出伪足,伪足的数量和长度随时间延长而逐渐增多和增长,胞质内的空泡也逐渐增加;24 h后,细胞表面积明显增大,伪足牵拉使细胞形状呈梭型。LPS处理后细胞上清中IL-6的含量较对照组显著增加(P<0.01)。流式细胞术检测发现细胞的前向角散射光(FSC)的数值(代表细胞大小)随LPS刺激时间的延长而增加,3和24 h分别增加了11.0%(P<0.05)和20.2%(P<0.01);侧向角散射光(SSC)的数值(代表表面颗粒数)也逐渐增加,3和24 h分别增加了21.6%(P<0.05)和68.0%(P<0.01)。结论流式细胞术可以定量检测小鼠腹腔巨噬细胞经LPS活化后的体积变化,为天然免疫细胞的活化研究提供了新的检测手段。

Objective To determine the volume change of mouse peritoneal macrophages after LPS-stimulation. Methods After stimulation with LPS for 3 and 24 hours, the cell morphological changes were observed under phase-con- trast microscope, the content of IL-6 in the cell supernatant was measured with ELISA （enzyme linked immunosorbent assay）, and the volume changes were analyzed with flow cytometry, respectively. Results In normal condition, mouse peritoneal macrophages are round with smooth edge and without vacuoles. After stimulated by LPS, the surface area of mouse peritoneal macrophages become larger than before. Pseudopedium and vacuoles can also be seen and as stimulation time extended, their number increased. Especially after 24 hours, mouse peritoneal macrophages turned into fusiform, and their pseudopodia fused with each other. After LPS stimulation, the content of IL-6 in the cell supernatant dramatically increased, illustrating the secretory pathway of inflammatory factor has been activated by LPS and the peritoneal macrophages have been activated. Flow cytometry observed increasing FSC （demonstrating cell size） by 11.0% （ P 〈 0. 05 ） after 3 hours and 20. 2% （ P 〈 0. 01 ） after 24 hours, as well as SSC （ demonstrating paticles in cell surface） increased by 21.6% （P 〈0. 05） after 3 hours and 68.0% （P 〈0. 01 ） after 24 hours. Conclusions Flow cytometry can determine the volume changes of mouse peritoneal macrophages quantitatively, providing new method for studying the activation and function of innate immunity cells.