Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.
Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.
复制标题
小鼠小胶质细胞的免疫磁富集和流式细胞仪表征。
DOI：
10.1016/j.jneumeth.2013.07.017
复制
发表时间：
2013-09-30
影响因子：
3
通讯作者：
Cox, Charles S.
中科院分区：
文献类型：
--
作者：
Bedi, Supinder S.;Smith, Philippa;Hetz, Robert A.;Xue, Hasen;Cox, Charles S.
CNS损伤后的炎症反应由小胶质细胞/巨噬细胞调节。M1经典活化的促炎细胞与M2替代活化的抗炎细胞的比率的变化揭示了免疫应答的方向。使用免疫组织化学通过形态学和细胞表面标志物对这些细胞进行常规鉴定和计数。我们使用受控皮质撞击（CCI）小鼠模型进行创伤性脑损伤（TBI），然后使用与CD 11b单克隆抗体缀合的磁珠从神经细胞悬液中分离小胶质细胞/巨噬细胞，以获得整个骨髓细胞群。通过M1表面标志物FcγRII/III和M2表面标志物CD 206的表达来评估CD 11b + CD 45 lo小胶质细胞的极化状态。TBI后，我们观察到与对侧相比，同侧半球的M1：M2比率增加，表明CCI后24小时，小胶质细胞极化发生转移，定位于损伤半球。开发和完善这些方法的主要动力是需要准确地量化小胶质细胞激活状态，而不依赖于连续免疫组化载玻片的手动形态计量计数。富集的细胞悬浮液的流式细胞术分析提供了对小胶质细胞极化状态的定量测量，其与现有方法互补，但用于整个细胞群体。总之，我们使用免疫磁珠从受伤的大脑中分离骨髓细胞，然后染色表面抗原，以流式细胞术鉴定和分类小胶质细胞为经典活化的M1或替代活化的M2，产生M1：M2细胞的比例，这在研究减少或重定向神经炎症的尝试中是有用的。
The inflammatory response after a CNS injury is regulated by microglia/macrophages. Changes in the ratio of M1 classically activated pro-inflammatory cells versus M2 alternatively activated anti-inflammatory cells reveal the direction of the immune response. These cells are routinely identified and enumerated by morphology and cell-surface markers using immunohistochemistry. We used a controlled cortical impact (CCI) mouse model for traumatic brain injury (TBI), then isolated microglia/macrophages from neural cell suspensions using magnetic beads conjugated to CD11b monoclonal antibody to obtain the entire myeloid population. Polarization states of CD11b+CD45lo microglia were evaluated by expression of M1 surface marker FcγRII/III and M2 surface marker CD206. After TBI, we observed an increase in M1:M2 ratio in the ipsilateral hemisphere when compared to the contralateral side, indicating that 24 hours after CCI, a shift in microglia polarization occurs localized to the hemisphere of injury. The major impetus for developing and refining the methods was the need to accurately quantify microglial activation states without reliance on manual morphometric counting of serial immunohistochemistry slides. Flow cytometric analysis of enriched cell suspensions provides quantitative measurement of microglial polarization states complementary to existing methods, but for entire populations of cells. In summary, we used immunomagnetic beads to isolate myeloid cells from injured brain, then stained surface antigens to flow cytometrically identify and categorize microglia as either classically activated M1 or alternatively activated M2, generating a ratio of M1:M2 cells which is useful in studying attempts to reduce or redirect neuroinflammation.
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影响因子：
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作者：
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通讯作者：
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