Pippin Jeffrey W, Loretz Carol J, Eng Diana G, Wessely Oliver, Shankland Stuart J
Division of Nephrology, University of Washington, Seattle, WA, USA.
Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA.
Bio Protoc. 2025 Jul 5;15(13):e5364. doi: 10.21769/BioProtoc.5364.
Glomerular diseases characterized by injury to post-mitotic epithelial cells called podocytes are a leading cause of chronic kidney disease. Yet, isolating podocytes from the kidney for transcriptomic, proteomic, and metabolomic studies has been a major technical challenge. Protocols utilizing glomerular sieving and laser capture methods are of limited use because they are not podocyte-specific but instead capture all four glomerular cell types. Here, we present a magnetic-activated cell sorting (MACS) method where podocytes are isolated from digested whole kidneys using antibodies specific to extracellular antigens on podocytes. Using microbeaded secondary antibodies binding to the podocyte-specific primary antibodies allows sorting of the podocytes using a magnet. This podocyte-only cell fraction is a unique source of in vivo-derived cells for molecular and cellular experiments. Key features • The protocol isolates a podocyte-only cell population from kidneys that is readily available for molecular and cellular studies. • The non-podocyte fraction serves as a matching negative control. • High cell yields are obtained. • The method can be applied to separately isolate podocytes from the outer cortex and juxtamedullary regions of the kidney.
以有丝分裂后上皮细胞（即足细胞）损伤为特征的肾小球疾病是慢性肾脏病的主要病因。然而，从肾脏中分离足细胞用于转录组学、蛋白质组学和代谢组学研究一直是一项重大技术挑战。利用肾小球筛分和激光捕获方法的方案用途有限，因为它们并非足细胞特异性的，而是会捕获所有四种肾小球细胞类型。在此，我们介绍一种磁激活细胞分选（MACS）方法，即使用针对足细胞细胞外抗原的特异性抗体从消化后的全肾中分离足细胞。使用与足细胞特异性一抗结合的微珠二抗，可通过磁体对足细胞进行分选。这种仅含足细胞的细胞组分是用于分子和细胞实验的体内来源细胞的独特来源。关键特性 • 该方案可从肾脏中分离出仅含足细胞的细胞群体，便于进行分子和细胞研究。 • 非足细胞组分可作为匹配的阴性对照。 • 可获得高细胞产量。 • 该方法可用于分别从肾脏的外皮质和近髓区域分离足细胞。
