Isolation of avian osteoclasts: improved techniques to preferentially purify viable cells.
Isolation of avian osteoclasts: improved techniques to preferentially purify viable cells.
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禽类破骨细胞的分离：改进技术以优先纯化活细胞。
DOI：
10.1002/jbmr.5650060409
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Osdoby,P
中科院分区：
文献类型：
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作者：
Oursler,MJ;Collin-Osdoby,P;Anderson,F;Li,L;Webber,D;Osdoby,P
在用于从各种物种中获取和研究分离的破骨细胞的许多不同方法中，低钙饮食的产蛋母鸡已被证明是相对大量破骨细胞的最丰富来源之一。然而，最近的报告和我们自己的观察表明，通过这种方法收获的破骨细胞中只有很小一部分是有活力的。（1）获得大量有活力的破骨细胞的困难限制了破骨细胞功能和调节的研究，因此寻求新的分离方法。本报告描述了一种破骨细胞分离程序，旨在大幅富集大量可行的真实破骨细胞。破骨细胞和污染单核细胞之间的大小和细胞密度差异已被用于开发破骨细胞富集方法。连续的非酶和酶程序，然后进行细胞密度分离，已经产生了三个破骨细胞群，这些破骨细胞源自维持低钙饮食的雏鸡。使用破骨细胞导向的单克隆抗体 121F 监测到连续分离方案期间骨相关破骨细胞的相应减少。前两个群体含有 40% 的破骨细胞，主要是 (>99%) 无法存活，但第三个群体包含的存活破骨细胞多出 8 倍，与前两个群体相比，有效地将存活破骨细胞的比例增加了 25 倍以上。分离的活细胞群 3 细胞的破骨细胞样性质是通过证明褶皱边界形成、拥有 121F 单克隆抗体反应性破骨细胞抗原、骨颗粒吸收活性以及通过透射和扫描电子显微镜显示的皮质骨切片上的吸收坑形成来确定的。
Among the many different methods that have been used to obtain and study isolated osteoclasts from a variety of species, the egg‐laying hen maintained on a low‐calcium diet has proven to be one of the richest sources of relatively large numbers of osteoclasts. However, recent reports and our own observations indicate that only a very small proportion of the osteoclasts harvested by such methods are viable.(1)The difficulty in obtaining large numbers of viable osteoclasts has restricted studies of osteoclast function and regulation, and so new isolation methods were sought. This report describes an osteoclast isolation procedure designed to substantially enrich for large numbers of viable authentic osteoclasts. Size and cell density differences between osteoclasts and contaminating mononuclear cells have been exploited in developing the methods for osteoclast enrichment. Sequential nonenzymatic and enzymatic procedures, followed by cell density separations, have yielded three populations of osteoclasts derived from chick hatchlings maintained on a low‐calcium diet. A corresponding decrease in bone‐associated osteoclasts during the sequential isolation scheme has been monitored using an osteoclast‐directed monoclonal antibody, 121F. The first two populations contain 40% osteoclasts, which are predominantly (>99%) nonviable, but the third population contains 8‐fold more viable osteoclasts, effectively increasing the proportion of viable osteoclasts more than 25‐fold in comparison with the first two populations. The osteoclast‐like nature of the isolated viable population 3 cells was established by demonstrating ruffled border formation, possession of the 121F monoclonal antibody‐reactive osteoclast antigen, bone particle resorption activity, and resorption pit formation on cortical bone slices revealed by transmission and scanning electron microscopy.