Identification of Myeloid Cell Subsets in Murine Lungs Using Flow Cytometry
Identification of Myeloid Cell Subsets in Murine Lungs Using Flow Cytometry
复制标题
DOI：
10.1165/rcmb.2012-0366ma
复制
发表时间：
2013-08-01
影响因子：
6.4
通讯作者：
Blackwell, Timothy S.
中科院分区：
文献类型：
--
作者：
Zaynagetdinov, Rinat;Sherrill, Taylor P.;Blackwell, Timothy S.
尽管细胞表面标志物的基于抗体的识别已被广泛用于免疫细胞的鉴定，但不同细胞类型的标志物表达的重叠和抗体组的不一致使用导致骨髓细胞亚群缺乏明确定义的特征。我们开发了一种10-荧光染料流式细胞术面板，用于鉴定和定量肺中的髓样细胞，包括肺单核细胞、髓样树突状细胞、肺泡和间质巨噬细胞以及中性粒细胞。在对存活的CD 45（+）白细胞进行初步分选后，我们检测了基于CD 68表达的三种白细胞亚群：CD 68（-）、CD 68（低）和CD 68（高）。对CD 68 hi群体的进一步表征显示了CD 45（+）/CD 68（hi）/F4/80（+）/CD 11b（-）/CD 11 c（+）/Gr 1（-）肺泡巨噬细胞和CD 45（+）/CD 68（hi）/F4/80（-）/CD 11 c（+）/Gr 1（-）/CD 103（+）/主要组织相容性复合物（MHC）II类hi树突状细胞。CD 68（低）群体主要包含CD 45（+）/CD 68（低）/F4/80（+）/CD 11b（+）/CD 11 c（+）/Gr 1（-）/CD 14（低）间质巨噬细胞和CD 45（+）/CD 68（低）/F4/80（+）/CD 11b（+）/CD 11 c（-）/Gr 1（低）/CD 14（hi）单核细胞，而CD 68 2群体包含中性粒细胞（CD 45（+）/CD 68（-）/F4/80（-）/CD 11b（+）/Gr 1（hi））。通过FACS分选细胞的形态学分析、功能研究和使用脂质体氯膦酸盐消耗特定巨噬细胞亚群证实了细胞特征的有效性。我们相信，我们的方法提供了一个准确的和可重复的方法，用于分离，定量和表征骨髓细胞亚群在肺，这可能是有用的研究骨髓细胞在各种病理过程中的作用。
Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD45(+) leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68(-), CD68(low), and CD68(hi). Further characterization of the CD68 hi population revealed CD45(+)/CD68(hi)/F4/80(+)/CD11b(-)/CD11c(+)/Gr1(-) alveolar macrophages and CD45(+)/CD68(hi)/F4/80(-)/CD11c(+)/Gr1(-)/CD103(+)/major histocompatibility complex (MHC) class IIhi dendritic cells. The CD68(low) population contained primarily CD45(+)/CD68(low)/F4/80(+)/CD11b(+)/CD11c(+)/Gr1(-)/CD14(low) interstitial macrophages and CD45(+)/CD68(low)/F4/80(+)/CD11b(+)/CD11c(-)/Gr1(low)/CD14(hi) monocytes, whereas the CD68 2 population contained neutrophils (CD45(+)/CD68(-)/F4/80(-)/CD11b(+)/Gr1(hi)). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.