TY - JOUR
T1 - Identification of myeloid cell subsets in murine lungs using flow cytometry
AU - Zaynagetdinov, Rinat
AU - Sherrill, Taylor P.
AU - Kendall, Peggy L.
AU - Segal, Brahm H.
AU - Weller, Kevin P.
AU - Tighe, Robert M.
AU - Blackwell, Timothy S.
PY - 2013/8
Y1 - 2013/8
N2 - Although the antibody-basedrecognition 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-, CD68low, and CD68hi. Further characterization of the CD68hi population revealed CD45+/CD68hi/F4/ 80+/CD11b-/CD11c+/Gr1- alveolar macrophages and CD45+/CD68hi/F4/80-/CD11c +/Gr1-/CD103+/major histocompatibility complex (MHC) class IIhi dendritic cells. The CD68low population contained primarily CD45+/CD68low/F4/80 +/CD11b+/CD11c+/ Gr1-/CD14 low interstitial macrophages and CD45+/CD68 low/F4/ 80+/CD11b+/CD11c-/Gr1 low/CD14hi monocytes, whereas the CD68- population contained neutrophils (CD45+/CD68-/F4/ 80 -/CD11b+/Gr1hi). The validity of cellular signatureswas 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.
AB - Although the antibody-basedrecognition 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-, CD68low, and CD68hi. Further characterization of the CD68hi population revealed CD45+/CD68hi/F4/ 80+/CD11b-/CD11c+/Gr1- alveolar macrophages and CD45+/CD68hi/F4/80-/CD11c +/Gr1-/CD103+/major histocompatibility complex (MHC) class IIhi dendritic cells. The CD68low population contained primarily CD45+/CD68low/F4/80 +/CD11b+/CD11c+/ Gr1-/CD14 low interstitial macrophages and CD45+/CD68 low/F4/ 80+/CD11b+/CD11c-/Gr1 low/CD14hi monocytes, whereas the CD68- population contained neutrophils (CD45+/CD68-/F4/ 80 -/CD11b+/Gr1hi). The validity of cellular signatureswas 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.
KW - Alveolar macrophages
KW - Interstitial macrophages
KW - Monocytes
KW - Myeloid dendritic cells
KW - Neutrophils
UR - http://www.scopus.com/inward/record.url?scp=84881492566&partnerID=8YFLogxK
U2 - 10.1165/rcmb.2012-0366MA
DO - 10.1165/rcmb.2012-0366MA
M3 - Article
C2 - 23492192
AN - SCOPUS:84881492566
SN - 1044-1549
VL - 49
SP - 180
EP - 189
JO - American Journal of Respiratory Cell and Molecular Biology
JF - American Journal of Respiratory Cell and Molecular Biology
IS - 2
ER -