A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation
A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation
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DOI：
10.1016/s0002-9440(10)61719-1
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发表时间：
2001-08-01
影响因子：
6
通讯作者：
Ridger, VC
中科院分区：
文献类型：
--
作者：
Cotter, MJ;Norman, KE;Ridger, VC
不适当的中性粒细胞激活与几种临床上重要的炎症状态的病理有关。虽然小鼠模型被广泛用于研究这种病理过程，但从小鼠血液中分离出存活的、静止的中性粒细胞的可靠方法还没有开发出来。在这里，我们描述了一种基于负免疫磁分离的新方法，该方法产生高纯度的小鼠中性粒细胞群体。血液与不想要的细胞上的特定细胞标志物的抗体混合，然后与二次抗体包裹的磁珠孵化。在磁场内的柱子中运行该制剂后，标记的细胞被保留，并收集富含中性粒细胞的流出物。这种方法产生95%的活性中性粒细胞的纯悬液，97%的活性中性粒细胞，相当于从全血中恢复70%的中性粒细胞。流式细胞仪分析显示，分离的中性粒细胞表面L-选择素和CD18的表达与全血中性粒细胞相比差异不大，表明中性粒细胞在分离过程中被激活的程度最低。佛波醇12-肉豆蔻酸13-醋酸酯刺激后，L选择素表达减少，CD18表达增加。分离的中性粒细胞在琼脂糖凝胶下迁移，对fMLP的反应，以及输入受体小鼠的荧光标记的中性粒细胞以类似于内源性白细胞的方式与毛细血管后小静脉相互作用。这些发现表明，用这种方法分离的中性粒细胞可以用于体外和体内的炎症研究。
Inappropriate neutrophil activation has been implicated in the pathology of several clinically important inflammatory conditions. Although murine models are extensively used in the investigation of such pathological processes, a reliable method by which viable, quiescent neutrophils; can be isolated from murine blood has not been developed. Here we describe a novel method based on negative immunomagnetic separation, which yields highly pure populations of murine neutrophils. Blood is incubated with a cocktail of antibodies against specific cell markers on unwanted cells, and then with secondary antibody-coated magnetic beads. After running the preparation through a column within a magnetic field, labeled cells are retained, and a neutrophil-rich effluent is collected. This method yields a >95% pure suspension of >97% viable neutrophils, recovering similar to 70% of neutrophils from whole blood. Flow cytometric analysis shows little difference in surface L-selectin and CD18 expression on isolated neutrophils compared with neutrophils in whole blood, indicating that neutrophils are minimally activated by the isolation process. Stimulation with phorbol 12-myristate 13-acetate (PMA) reduced L-selectin and increased CD18 expression. isolated neutrophils migrate under agarose in response to fMLP, and fluorescently labeled neutrophils transfused into recipient mice interact with postcapillary venules in a manner comparable to endogenous leukocytes. These findings show that neutrophils isolated using this method can be used for inflammatory studies in vitro and in vivo.