Centre for Genetics, Ecology & Physiology, School of Health and Sport Sciences, University of the Sunshine Coast, QLD, Australia; VasoActive Research Group, School of Health and Sport Sciences, University of the Sunshine Coast, QLD, Australia.
VasoActive Research Group, School of Health and Sport Sciences, University of the Sunshine Coast, QLD, Australia.
J Immunol Methods. 2019 Sep;472:75-78. doi: 10.1016/j.jim.2019.04.005. Epub 2019 Jun 20.
Innate immune cell defects contribute to severe autoimmunity and the pathogenesis of inflammatory disease. Monocyte-derived macrophages typically retain disease related signatures and represent an excellent in vitro model to uncover and validate mechanisms contributing to specific pathological states. Monocyte isolation procedures vary widely in terms of purity, yield, cost, degree of technical difficulty and volume of peripheral blood needed. This paper outlines a novel isolation method that yields monocytes through density gradient centrifugation (Ficoll® and hyperosmotic Percoll®). The protocol has been optimised for small volumes of blood (42 ml) and is simple, reproducible and inexpensive compared to other methods. Monocyte recovery is 70% (relative to monocyte numbers within the buffy coat) and the highly functional macrophages produced are characterised by excellent purity (98.6 ± 0.6%) and intact activation and phagocytic capacities. The method is well suited to investigations involving patient populations where a particular subset of immune cells is known to contribute to the pathogenesis of a specific disease or is aberrant as a consequence of that disease.
先天免疫细胞缺陷导致严重的自身免疫和炎症性疾病的发病机制。单核细胞衍生的巨噬细胞通常保留与疾病相关的特征，是揭示和验证导致特定病理状态的机制的极佳体外模型。单核细胞的分离程序在纯度、产量、成本、技术难度和所需外周血体积方面差异很大。本文概述了一种通过密度梯度离心（Ficoll® 和高渗 Percoll®）分离单核细胞的新方法。该方案已针对小体积血液（42ml）进行了优化，与其他方法相比，该方案简单、可重复且成本低廉。单核细胞的回收率为 70%（相对于血涂片层中的单核细胞数量），所产生的功能强大的巨噬细胞具有极好的纯度（98.6±0.6%）和完整的激活和吞噬能力。该方法非常适合涉及患者群体的研究，其中已知特定免疫细胞亚群有助于特定疾病的发病机制，或者由于该疾病而异常。
