A simplified method for isolation of large numbers of defined nephron segments
A simplified method for isolation of large numbers of defined nephron segments
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DOI：
10.1152/ajprenal.1997.273.4.f650
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发表时间：
1997-10-01
影响因子：
4.2
通讯作者：
Schlatter, E
中科院分区：
文献类型：
--
作者：
Schafer, JA;Watkins, ML;Schlatter, E
我们描述了一种从大鼠和兔肾脏中分离大量肾单位片段的简化方法。与大多数以前的方案相比，肾脏没有灌注。从动物身上取出后，肾脏被切成碎片，随后在 37 摄氏度下在含有 0.5 mg/ml 胶原酶的培养基中消化。如果仅非常轻微且不频繁地搅拌制剂，则组织会逐渐分解成含有长肾单位碎片的悬浮液，通常由多个连接的片段组成。这些很容易被分类为同质片段群体，可用于酶测定、免疫印迹的蛋白质提取和逆转录聚合酶链式反应的 RNA 提取，所有这些都已在我们的实验室成功完成。为了进行比较，我们还检查了通过先前描述的更严格的方案制备的皮质集合管片段和细胞（E. Schlatter，U. Frobe，和 R. Greger. Pflugers Arch. 421：381-387，1992）。即使在无 Ca2+ 培养基中分离单细胞后，细胞仍保持其正常结构以及顶膜和基底外侧膜的明显分离。
We describe a simplified method for the isolation of large numbers of nephron segments from rat and rabbit kidneys. In contrast to most previous protocols, the kidneys are not perfused. After removal from the animal, the kidney is sliced and torn in pieces that are subsequently digested in culture medium containing 0.5 mg/ml of collagenase at 37 degrees C. If the preparation is agitated only very gently and infrequently, then the tissue gradually falls apart-into a suspension containing long nephron fragments, often consisting of multiple connected segments. These are easily sorted into homogeneous segment populations that can be used for enzyme assays, protein extraction for immunoblotting, and RNA extraction for reverse transcription-polymerase chain reaction, all of which have been done successfully in our laboratory. For comparison, we have also examined cortical collecting tubule segments and cells prepared by the more rigorous protocol described previously (E. Schlatter, U. Frobe, and R. Greger. Pflugers Arch. 421: 381-387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells maintain their normal architecture and a distinct separation of apical and basolateral membranes.