Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.
Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.
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DOI：
10.1186/1742-2094-9-147
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发表时间：
2012-06-28
影响因子：
9.3
通讯作者：
Watters JJ
中科院分区：
文献类型：
--
作者：
Nikodemova M;Watters JJ
小胶质细胞活化在与几乎所有CNS疾病相关的神经炎症中起关键作用，尽管它们在正常CNS生理学中的作用越来越受到重视。通常通过分析CNS组织匀浆中的促炎介质来评估神经炎症，假设小胶质细胞是这些分子的主要来源。然而，CNS中的其他细胞类型也可以合成炎性分子。因此，为了能够离体直接分析小胶质细胞的活性，需要一种有效、可靠和可重复的小胶质细胞分离方法。在酶消化脑组织和髓鞘去除后，使用免疫磁性分离CD 11b+细胞，得到高度纯化的小胶质细胞，没有星形胶质细胞或神经元污染。我们使用了三种髓鞘去除方法（30%Percoll，0.9 mol/l蔗糖和抗髓鞘磁珠），并比较了它们对小胶质细胞活力和产量的影响。为了确定分离过程本身是否激活小胶质细胞，我们使用流式细胞术检查脑组织匀浆中的小胶质细胞特性，并从对照组和脂多糖（LPS）处理的小鼠中分离小胶质细胞。该方法产生了高度纯化的CD 11b+细胞群，其特性反映了其体内表型。髓鞘去除方法显著影响分离细胞的活力和产量。虽然小胶质细胞表型在所有使用的方法中相当，但Percoll获得的CD 11b+细胞的活力和数量最高。从LPS处理的小鼠中分离的小胶质细胞显示出促炎表型，如通过TNF-α水平上调所确定的，而从对照小鼠中分离的小胶质细胞则没有。免疫磁性分离是一种有效的方法，从中枢神经系统分离小胶质细胞，并同样适用于分离静止和活化的小胶质细胞。该技术允许离体评价小胶质细胞活性，其准确地反映了它们在体内的活性。通过该方法获得的小胶质细胞可用于多种下游应用，包括qRT-PCR、ELISA、蛋白质印迹和流式细胞术，以分析任何数量的CNS病理或损伤中的小胶质细胞活性。
Microglial activation plays a key role in the neuroinflammation associated with virtually all CNS disorders, although their role in normal CNS physiology is becoming increasingly appreciated. Neuroinflammation is often assessed by analyzing pro-inflammatory mediators in CNS tissue homogenates, under the assumption that microglia are the main source of these molecules. However, other cell types in the CNS can also synthesize inflammatory molecules. Hence, to enable direct analysis of microglial activities ex vivo, an efficient, reliable, and reproducible method of microglial isolation is needed. After enzymatic digestion of brain tissues and myelin removal, CD11b+ cells were isolated using immunomagnetic separation, yielding highly purified microglia without astrocyte or neuronal contamination. We used three methods of myelin removal (30% Percoll, 0.9 mol/l sucrose and anti-myelin magnetic beads), and compared their effects on microglial viability and yield. To determine whether the isolation procedure itself activates microglia, we used flow cytometry to examine microglial properties in brain-tissue homogenates and isolated microglia from control and lipopolysaccharide (LPS) -treated mice. This method yielded a highly purified CD11b+ cell population with properties that reflected their in vivo phenotype. The viability and yield of isolated cells were significantly affected by the myelin removal method. Although the microglial phenotype was comparable in all methods used, the highest viability and number of CD11b+ cells was obtained with Percoll. Microglia isolated from LPS-treated mice displayed a pro-inflammatory phenotype as determined by upregulated levels of TNF-α, whereas microglia isolated from control mice did not. Immunomagnetic separation is an efficient method to isolate microglia from the CNS, and is equally suitable for isolating quiescent and activated microglia. This technique allows evaluation of microglial activities ex vivo, which accurately reflects their activities in vivo. Microglia obtained by this method can be used for multiple downstream applications including qRT-PCR, ELISA, Western blotting, and flow cytometry to analyze microglial activities in any number of CNS pathologies or injuries.
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影响因子：
3
作者：
Marek, Ryan;Caruso, Michael;Sarma, Jayasri Das
通讯作者：
Sarma, Jayasri Das
影响因子：
56.9
作者：
Nimmerjahn, A;Kirchhoff, F;Helmchen, F
通讯作者：
Helmchen, F
影响因子：
5.3
作者：
Jimenez, Sebastian;Baglietto-Vargas, David;Vitorica, Javier
通讯作者：
Vitorica, Javier
影响因子：
4.2
作者：
Burkert, Kristina;Moodley, Kiebashne;Graham, E. Scott
通讯作者：
Graham, E. Scott
影响因子：
4.7
作者：
Festoff, Barry W.;Ameenuddin, Syed;Citron, Bruce A.
通讯作者：
Citron, Bruce A.