Swamydas Muthulekha, Lionakis Michail S
Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, USA.
J Vis Exp. 2013 Jul 10(77):e50586. doi: 10.3791/50586.
Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.
中性粒细胞是先天性免疫系统的关键效应细胞。它们在急性炎症部位迅速募集，并根据炎症环境发挥保护或致病作用。尽管如此，尽管中性粒细胞在免疫中起着不可或缺的作用，但对于介导中性粒细胞在不同传染病和炎症条件下的效应和免疫致病作用的分子因素，仍缺乏详细的了解，部分原因是它们的半衰期短、处理这些细胞存在困难，以及缺乏可靠的实验方案来获得足够数量的中性粒细胞用于下游功能研究和过继转移实验。因此，非常需要简单、快速、经济且可靠的方法来收获足够数量的小鼠中性粒细胞，以评估诸如吞噬作用、杀伤作用、细胞因子产生、脱颗粒和迁移等功能。为此，我们提出了一种基于密度梯度离心的可重复方案，该方案可在任何实验室中进行调整，以从小鼠骨髓中分离出大量高纯度和高活力的中性粒细胞。此外，我们还提出了一种简单的方案，即使用细胞追踪染料标记分离出的中性粒细胞，然后将其过继转移到受体小鼠体内，并使用流式细胞术在转移后至少4小时内在多个组织中进行追踪。使用这种方法，可以成功地用不同的细胞追踪染料对野生型和基因缺陷型小鼠的中性粒细胞进行差异标记，以进行竞争性再填充研究，从而评估特定基因在体内中性粒细胞从血液向靶组织迁移中的直接作用。
