School of Life Sciences, South China Normal University.
School of Life Sciences, Guangzhou University;
J Vis Exp. 2024 Sep 27(211). doi: 10.3791/67315.
Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show that the microglial surface expresses potassium channels that regulate microglial activation, while abnormalities in these potassium channels can lead to neural diseases. Currently, whole-cell patch-clamp recordings of microglia are mostly performed on cultured primary microglia from fetal or newborn mice due to difficulties in conducting electrophysiological evaluations on acutely isolated microglia. This study introduces an easy-to-follow protocol for isolating hippocampal microglia from adult mice and performing whole-cell patch-clamp recordings on the isolated cells. Briefly, the brain was removed from a mouse after decapitation, the hippocampus was dissected bilaterally, and microglia were isolated using an adult mouse brain dissociation kit. The microglia were then purified using a magnetic-activated cell sorting (MACS) method and seeded onto coverslips. Successful microglial isolation was confirmed by immunofluorescent staining with anti-CD11 and anti-Iba1 antibodies. A cover slip was placed in a recording chamber, and the whole-cell potassium currents of the acutely isolated microglia were recorded under voltage-clamp conditions.
小胶质细胞是大脑中常驻的免疫细胞，与神经元相互作用以维持中枢神经系统 (CNS) 的稳态。研究表明，小胶质细胞表面表达的钾通道调节小胶质细胞的激活，而这些钾通道的异常可导致神经疾病。目前，由于难以对急性分离的小胶质细胞进行电生理评估，大多数全细胞膜片钳记录都是在培养的胎鼠或新生鼠来源的原代小胶质细胞上进行的。本研究介绍了一种从成年小鼠中分离海马小胶质细胞并对分离细胞进行全细胞膜片钳记录的简单易行的方案。简要地说，断头后从小鼠脑中取出大脑，双侧解剖出海马体，使用成年小鼠脑解离试剂盒分离小胶质细胞。然后使用磁激活细胞分选 (MACS) 方法对小胶质细胞进行纯化，并接种到盖玻片上。用抗 CD11 和抗 Iba1 抗体进行免疫荧光染色来确认小胶质细胞的成功分离。将盖玻片放置在记录室中，在电压钳条件下记录急性分离的小胶质细胞的全细胞钾电流。
