Department of Medicine, McMaster University & St Joseph's Healthcare, Hamilton, ON, Canada.
Department of Medicine, Alberta Respiratory Centre, University of Alberta, Edmonton, AB, Canada.
Methods Mol Biol. 2021;2241:15-25. doi: 10.1007/978-1-0716-1095-4_2.
The choice of isolation technique for human peripheral blood eosinophils contributes to the understanding of clinically relevant data derived from in vitro research. Since the 1990s, eosinophils have been conventionally isolated via density gradient centrifugation followed by negative immunomagnetic selection using anti-CD16 antibody-coated magnetic beads. Due to recent advancements in molecular techniques, "newer" methods have been made commercially available that drastically reduce user handling and processing time while maintaining high population purity. Here, we describe an isolation procedure using one of these methods, the human MACSxpress Whole Blood Isolation Kit, as well as outline protocols for differential staining and flow cytometry analysis to evaluate the purity and activation state of isolated cells. In addition, we highlight an in vitro degranulation assay that may be used to verify the intact functionality of the isolated eosinophils.
选择用于分离人外周血嗜酸性粒细胞的技术有助于理解体外研究中获得的与临床相关的数据。自 20 世纪 90 年代以来，嗜酸性粒细胞通常通过密度梯度离心分离，然后使用抗 CD16 抗体包被的磁珠进行负免疫磁选。由于分子技术的最新进展，已经商业化提供了“更新”的方法，这些方法大大减少了用户的处理和加工时间，同时保持了高群体纯度。在这里，我们描述了一种使用其中一种方法的分离程序，即人类 MACSxpress 全血分离试剂盒，并概述了用于差异染色和流式细胞术分析的方案，以评估分离细胞的纯度和激活状态。此外，我们还强调了一种体外脱颗粒测定，可用于验证分离的嗜酸性粒细胞的完整功能。
