Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium
Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium
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DOI：
10.3791/53035
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发表时间：
2015-09-01
影响因子：
1.2
通讯作者：
Staruschenko, Alexander
中科院分区：
文献类型：
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作者：
Pavlov, Tengis S.;Ilatovskaya, Daria V.;Staruschenko, Alexander
多囊肾病期间囊肿的形成和扩张是一个复杂的过程，其特征是肾小管细胞增殖、管腔液积聚和细胞外基质形成异常。离子通道的活性和细胞内钙信号传导是决定肾小管上皮功能的关键生理参数。我们开发了一种适用于通过膜片钳技术实时观察离子通道活性并记录从肾囊肿中新鲜分离的单层上皮细胞内 Ca2+ 水平的方法。 PCK 大鼠是常染色体隐性多囊肾病 (ARPKD) 的遗传模型，用于离子通道和钙通量的离体分析。这里描述的是一个详细的分步程序，旨在从 PCK 或正常 Sprague Dawley (SD) 大鼠中分离囊性单层和非扩张小管，并监测单通道活动和细胞内 Ca2+ 动态。该方法不需要酶处理，并且允许在新鲜分离的上皮单层的天然环境中进行分析。此外，该技术对细胞内钙的变化非常敏感，并生成高分辨率图像以进行精确测量。最后，分离的囊性上皮可进一步用于抗体或染料染色、原代培养物的制备和各种生化测定的纯化。
Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.