Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights

Cytometry A. 2004 Oct;61(2):170-77. doi: 10.1002/cyto.a.20064.


Background: The need to accurately discriminate dendritic cells (DCs) and macrophages (Mphs) in mouse lungs is critical given important biological differences. However, a validated flow cytometry-based method is still lacking, resulting in much confusion between both cell types.

Methods: Single-cell suspensions freshly obtained from collagenase-digested lung tissue were stained with a CD11c-specific monoclonal antibody, detected using a PE-Cy5 or APC-conjugated secondary reagent. Cellular immunophenotype was simultaneously explored using a panel of PE-conjugated markers. The FL1 or FITC-detection channel was reserved for the assessment of autofluorescence.

Results: CD11c-bright cells were heterogeneous and displayed a bimodal distribution with regard to autofluorescence (AF). CD11c+/low-AF cells were lineage-negative and showed features compatible with myeloid DCs. This was confirmed by morphology, potent T-cell stimulatory function in a mixed-leukocyte reaction, surface expression of MHCII and costimulatory molecules, and further immunophenotypical criteria, including the expression of Mac-1 and absence of CD8alpha. In contrast, CD11c+/high-AF cells displayed the features of pulmonary Mphs, including typical Mph morphology, very weak induction of T-cell proliferation, low to absent expression of MHCII and costimulatory molecules, and very low levels of Mac-1 as well as F4/80. We also show that only CD11c+/high-AF cells strongly expressed the macrophage marker MOMA-2, while interestingly Mac-3 was expressed at high levels by CD11c+/high-AF and low-AF alike.

Conclusions: This study shows that the combination of CD11c-expression and autofluorescence is necessary and sufficient to accurately separate DCs from macrophage subpopulations in mouse lungs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antigens, Differentiation / biosynthesis
  • CD11c Antigen / biosynthesis
  • CD8 Antigens / biosynthesis
  • Collagenases / metabolism
  • Dendritic Cells / cytology*
  • Flow Cytometry / instrumentation
  • Flow Cytometry / methods*
  • Immunophenotyping
  • Leukocytes / cytology
  • Lung / cytology
  • Macrophages / cytology*
  • Macrophages / immunology
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence
  • T-Lymphocytes / immunology


  • Antibodies, Monoclonal
  • Antigens, Differentiation
  • CD11c Antigen
  • CD8 Antigens
  • monocyte-macrophage differentiation antigen
  • Collagenases