Vander Top Elizabeth A, Perry Greg A, Gentry-Nielsen Martha J
Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA.
BMC Microbiol. 2006 Jul 12;6:61. doi: 10.1186/1471-2180-6-61.
Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs) isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS) to recruit PMNs to their lungs. They are then infected with live 5(-and 6) carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria.
The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides.
This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.
传统的吞噬作用检测是在体外使用从外周血或腹膜中分离出的多形核白细胞（PMN）以及经热灭活、预先调理过的生物体来进行的。这些检测可能无法充分模拟感染肺部的环境。因此，我们实验室开发了一种流式细胞术体内吞噬作用检测方法，该方法能够对大鼠肺部活细菌的PMN吞噬作用进行定量分析。在这些研究中，通过气管向大鼠注射脂多糖（LPS）以将PMN募集到肺部。然后用活的5（和6）羧基荧光素二乙酸琥珀酰亚胺酯（CFDA/SE）标记的3型肺炎链球菌感染它们。进行支气管肺泡灌洗，并使用针对每种细胞类型表面表位的单克隆抗体对驻留的肺泡巨噬细胞和募集的PMN进行标记。利用三色流式细胞术来识别细胞类型、定量募集情况并确定标记细菌的摄取情况。
从灌洗液中分离出的肺泡巨噬细胞和PMN的活力>95%。通过流式细胞术测量的灌洗液中PMN的百分比以及与CFSE标记的肺炎链球菌相关的PMN的百分比值与细胞涂片手动计数结果高度相关。
该检测方法适用于测量感染肺部内的细菌摄取情况。它可适用于其他生物体和/或动物模型系统。
