Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, IA 50011, USA.
J Neurosci Methods. 2011 Jan 15;194(2):287-96. doi: 10.1016/j.jneumeth.2010.11.001. Epub 2010 Nov 11.
Microglial cells play a dynamic role in the brain beyond their established function of immune surveillance. Activated microglia play key roles in neural development, neuroinflammation, neural repair and neurotoxicity. They are particularly important in several neurodegenerative diseases in which sustained microglial activation contributes to the progression of neurodegenerative processes. Consequently, understanding microglial function in CNS health and disease has become an area of active research in recent years. However, a significant obstacle to progress in this field has been the inherent difficulties in obtaining large amounts of primary microglial cells to routinely perform mechanistic studies and characterize signaling pathways regulating the dynamics of microglial activation. Herein, we describe a novel column-free magnetic separation protocol for high-yield isolation of primary microglia from mouse postnatal mixed glial cultures. The procedure is based on optimized culture conditions that enable high microglial cell densities in confluent mixed glial cultures followed by highly efficient recovery of pure microglia by magnetic separation. The novel column-free magnetic separation system utilizes tetrameric antibody complexes (TAC) with dual specificity for CD11b-PE labeled microglia and dextran magnetic nanoparticles. An FcR blocker (anti-CD16/32) is added to enhance the purity of the microglial separation by preventing non-specific labeling of other cell types. This procedure yields on average >3×10⁶ microglial cells per mouse pup, with a remarkable purity of 97% and recovery of around 87% of microglia from the mixed glial population. Importantly, the microglia obtained by this method are fully functional and respond like cells obtained by conventional isolation techniques.
小胶质细胞在大脑中的作用不仅仅局限于其免疫监视的传统功能。激活的小胶质细胞在神经发育、神经炎症、神经修复和神经毒性中发挥关键作用。它们在几种神经退行性疾病中尤为重要，其中持续的小胶质细胞激活导致神经退行性过程的进展。因此，近年来，理解小胶质细胞在中枢神经系统健康和疾病中的功能已成为一个活跃的研究领域。然而，该领域进展的一个重大障碍是，获得大量原代小胶质细胞以常规进行机制研究并表征调节小胶质细胞激活动力学的信号通路一直具有固有困难。在此，我们描述了一种从新生小鼠混合胶质培养物中高效分离原代小胶质细胞的新型无柱磁分离方案。该方案基于优化的培养条件，使混合胶质培养物中达到高的小胶质细胞密度，然后通过磁分离高效回收纯小胶质细胞。新型无柱磁分离系统利用四聚体抗体复合物（TAC）对 CD11b-PE 标记的小胶质细胞和葡聚糖磁纳米颗粒具有双重特异性。添加 FcR 阻断剂（抗-CD16/32）可通过防止其他细胞类型的非特异性标记来增强小胶质细胞分离的纯度。该方案平均每只幼鼠可获得 >3×10⁶个小胶质细胞，纯度显著达到 97%，且从混合胶质群体中回收的小胶质细胞约为 87%。重要的是，通过这种方法获得的小胶质细胞具有完全的功能，并且其反应类似于通过传统分离技术获得的细胞。
