An efficient method to isolate and culture mouse Kupffer cells
An efficient method to isolate and culture mouse Kupffer cells
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DOI：
10.1016/j.imlet.2013.12.002
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发表时间：
2014-03-01
影响因子：
4.4
通讯作者：
He, Kun
中科院分区：
文献类型：
--
作者：
Li, Pei-zhi;Li, Jin-zheng;He, Kun
枯否细胞（Kupffer cell，KCs）在肝脏的生理和病理功能中起着重要作用。虽然KCs的分离方法已被很好地描述，但大多数方法复杂且耗时。此外，这些方法主要用于分离人和大鼠的KCs。本研究采用酶组织处理、梯度离心和选择性贴壁三步法从小鼠肝脏中分离出足够数量和纯度的KCs。采用F4/80免疫荧光法和流式细胞术进行细胞鉴定。组合方法产生了令人满意的高产率，每个肝脏5-6 x 10（6）KC，F4/80阳性率超过92.0%，活细胞率为98.5%。培养24 h后，KCs呈典型的巨噬细胞形态特征，形态不规则，胞浆透明，核呈肾状。吞噬实验表明，分离的细胞具有较强的吞噬活性。我们分离的KC功能完整，并表现出LPS诱导的浓度依赖性TNF-α产生。该方法在不改变小鼠KCs功能的前提下，节省了大量的胶原酶和时间，是一种高纯度、高产量的KCs分离方法。(C)2013爱思唯尔有限公司版权所有。
Kupffer cells (KCs) play an essential role in the physiological and pathological functions of the liver. Although the isolation methods of KCs have been well-described, most of them are sophisticated and time-consuming. In addition, these methods are mainly used for isolating the KCs of the human and rat. In this study, a three-step procedure was applied to isolate KCs in sufficient number and purity from mouse liver, including the techniques of enzymatic tissue treatment, gradient centrifugation, and selective adherence. F4/80 immunofluorescence and flow cytometry were used for cell identification. The combination method resulted in a satisfactorily high yield of 5-6 x 10(6) KCs per liver, over 92.0% positive for F4/80 and 98.5% viable cells. After 24 h of culturing, the KCs showed typical macrophage morphologic features such as irregular shape, transparent cytoplasm and kidney-like nucleus. The phagocytic assay showed that the isolated cells exhibited strong phagocytosis activity. The KCs we isolated were functionally intact and exhibited a concentration dependent TNF-alpha production induced by LPS. The method we described is an effective method to isolate mouse KCs in high purity and yield, which consuming fewer collagenase and time without altering the functional capacity of the KCs. (C) 2013 Elsevier B.V. All rights reserved.