Biology
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密度梯度离心法测定微分品质的精子
Chapters
Summary November 29th, 2018
Please note that all translations are automatically generated.
Click here for the English version.
本文介绍了密度梯度离心技术的性能及其在精子生理研究中的应用。
Transcript
该方法有助于回答精子生理学领域关键问题，如精子质量的化学决定。这种技术的主要优点是能够按质量分离大量样品，以及与替代方法相比的适应性。虽然这种方法可以提供精子质量差异的见解，它也可以应用于其他样本，如混合细胞类型或具有不同密度的亚细胞成分。
首先，在两个独立的管中进行两个3毫升的佩尔科尔稀释。在干净的试管中，用2毫升PBS稀释1毫升精液样品。轻轻管它混合溶液。
将 3 毫升的较低密度 Percoll sollution 添加到无菌锥形管中。然后，小心移液器 3 毫升的高密度 Percoll 溶液下低密度 Percoll 溶液。在以 45 度角倾斜锥形管时，移液器在 Percoll 密度梯度上稀释精液样品的 3 毫升。
制备白管后，在1500 G时将两个管离心20分钟。确保离心后在浴缸里形成三层不同的精液。使用移液器收集三层精液，从顶层开始，然后是中间层，最后以管底的硬颗粒结束。
将每个层转移到无菌微离心管。用 1.5 毫升 PBS 稀释每个精液样品，并在 1500 G 下将样品离心 10 分钟。最后，从上流液中倒出，用运动缓冲液重建颗粒。
在此协议中，Percoll 密度梯度离心技术（PDGC）用于将精液样本分离为三个不同的层。精子在高密度溶液下方分离成高质量层，在高密度解决方案和低密度溶液之间分离成中等质量层，在低密度溶液之上分离成低质量层。精子质量的这些差异从生存能力、流动性和可读性的差异中得到证明。
在尝试此过程时，重要的是要记住样品需要被带到室温。PDGC 的成功取决于对梯度的精心准备以及对隔离层的仔细收集。这项技术开发后，为我们在精子生物研究领域的研究铺平了道路，以探索鸟类繁殖的生物标志物。
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