Objective To establish an optimized method for isolation and purification of microglia from aged rat brain tissue,and the phenotype of microglia was detected by flow cytometry.Methods With young rats(3 months old)as control,the brain tissues of aged rats were immediately processed into single cell suspensions by mechanical dissociation and enzymatic digestion using type Ⅳ collagenase.Microglia were isolated on Percoll gradients(30％-37％-70％).The cells were stained with fluorescence-labeled antibodies and the phenotype of microglia was detected by flow cytometry.Results This study developed a method that enzymatic digestion and mechanical disso-ciation combined with density gradient centrifugation.More single cells could be obtained by using this method.And the survival rate of cells was more than 90％.The flow cytometric analysis showed that the expression of M1 microglia marker CD86 and MHC Ⅱ increased(P＜0.01),and the expression of M2 microglia marker CD200R increased(P＜0.01)in aged rats compared with that in young rats.Conclusion The use of type Ⅳ collagenase and mechanical digestion combined with density gradient centrifugation is good for isolating and purifying microglia from adult and aged rat brain tissue.
关键词
衰老/神经炎症/小胶质细胞/分离纯化/流式细胞术/表面标志物
Key words
aging/neuroinflammation/isolation and purification/microglia/flow cytometry/surface marker
维普
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